An update for atypical haemolytic uraemic syndrome: diagnosis and treatment. A consensus document. Review

n e f r o l o g i a. 2 0 1 5;35(5):421–447

RevistadelaSociedadEspañoladeNefrología www . r e v i s t a n e f r o l o g i a . c o m

Review

An

update

for

atypical

haemolytic

uraemic

syndrome:

Diagnosis

and

treatment.

A

consensus

document

夽

Josep

M.

Campistol

,

Manuel

Arias

,

Gema

Ariceta

,

Miguel

Blasco

,

Laura

Espinosa

,

Mario

Espinosa

_,

_Josep

_M.

_Grinyó

_,

_Manuel

_Macía

_,

_Santiago

_Mendizábal

_,

Manuel

Praga

_,

_Elena

_Román

_,

_Roser

_Torra

_,

_Francisco

_Valdés

_,

_Ramón

_Vilalta

_,

Santiago

Rodríguez

de

Córdoba

b_{ServiciodeNefrología,HospitalUniversitarioMarquésdeValdecilla,Santander,Spain}

c_{ServiciodeNefrologíaPediátrica,HospitalUniversitariMaterno-InfantilValld’Hebrón,UniversidadAutónomadeBarcelona,Barcelona,} Spain

d_{ServiciodeNefrologíaPediátrica,HospitalLaPaz,Madrid,Spain} e_{ServiciodeNefrología,HospitalUniversitarioReinaSofía,Córdoba,Spain}

f_{ServiciodeNefrología,HospitalUniversitarideBellvitge,HospitaletdeLlobregat,Barcelona,Spain} g_{ServiciodeNefrología,HospitalVirgendelaCandelaria,SantaCruzdeTenerife,Spain}

h_{ServiciodeNefrologíaPediátrica,HospitalLaFe,Valencia,Spain}

i_{ServiciodeNefrología,HospitalUniversitario12deOctubre,Madrid,Spain} j_{EnfermedadesRenalesHereditarias,FundacióPuigvert,Barcelona,Spain} k_{ServiciodeNefrología,ComplejoHospitalarioACoru ˜na,ACoru ˜na,Spain}

l_{DepartamentodeMedicinaCelularyMolecular,CentrodeInvestigacionesBiológicas(CSIC),Madrid,Spain}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9April2015 Accepted3July2015

Availableonline3December2015

Keywords:

Atypicalhaemolyticuraemic syndrome

Eculizumab Complement

Thromboticmicroangiopathy

a

b

s

t

r

a

c

t

Haemolyticuraemicsyndrome(HUS)isaclinicalentitydefinedasthetriadofnonimmune haemolyticanaemia,thrombocytopenia,andacuterenalfailure,inwhichtheunderlying lesionsaremediatedbysystemicthromboticmicroangiopathy(TMA).Differentcausescan induce theTMAprocessthatcharacterisesHUS.Inthisdocumentweconsideratypical HUS (aHUS)a sub-typeofHUSin whichtheTMA phenomenaare theconsequenceof theendotelialdamageinthemicrovasculatureofthekidneysandotherorgansduetoa disregulationoftheactivityofthecomplementsystem.Inrecentyears,avarietyof aHUs-related mutationshavebeenidentifiedingenesofthecomplementsystem,whichcan explainapproximately60%oftheaHUScases,andanumberofmutationsand polymor-phismshavebeenfunctionallycharacterised.ThesefindingshavestablishedthataHUSisa consequenceoftheinsufficientregulationoftheactivationofthecomplementoncell sur-faces,leadingtoendotelialdamagemediatedbyC5andthecomplementterminalpathway.

夽

Pleasecitethisarticleas:CampistolJM,AriasM,AricetaG,BlascoM,EspinosaL,EspinosaM,etal.Actualizaciónensíndromehemolítico urémicoatípico:diagnósticoytratamiento.Documentodeconsenso.Nefrologia.2015;35:421–447.

∗ _{Correspondingauthor.}

E-mailaddress:JMCAMPIS@clinic.ub.es(J.M.Campistol).

2013-2514/©2015SociedadEspa ˜noladeNefrología.PublishedbyElsevierEspaña,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

EculizumabisamonoclonalantibodythatinhibitstheactivationofC5andblocksthe gen-erationofthepro-inflammatorymoleculeC5aandtheformationofthecellmembrane attackcomplex.InprospectivestudiesinpatientswithaHUS,theuseofEculizumabhas shownafastandsustainedinterruptionoftheTMAprocessandithasbeenassociated withsignificativelong-termimprovementsinrenalfunction,theinterruptionofplasma therapyandimportantreductionsintheneedofdialysis.Accordingtotheexistingliterature andtheaccumulatedclinicalexperience,theSpanishaHUSGrouppublishedaconsensus documentwithrecommendationsforthetreatmentofaHUs(Nefrologia2013;33[1]:27–45). In the current online versionof this document, weupdate the aetiological classifica-tionofTMAs,thepathophysiologyofaHUS,itsdifferentialdiagnosisanditstherapeutic management.

©2015SociedadEspa ˜noladeNefrología.PublishedbyElsevierEspaña,S.L.U.Thisisan openaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Actualización

en

síndrome

hemolítico

urémico

atípico:

diagnóstico

y

tratamiento.

Documento

de

consenso

Palabrasclave:

Síndromehemolíticourémico atípico

Eculizumab Complemento

Microangiopatíatrombótica

r

e

s

u

m

e

n

Elsíndromehemolíticourémico(SHU)esunaentidadclínicadefinidaporlatríada ane-mia hemolíticano inmune,trombocitopenia einsuficienciarenal aguda, enlaquelas lesionessubyacentesestánmediadasporunprocesodemicroangiopatíatrombótica(MAT) sistémico. Distintascausaspueden desencadenarelprocesodeMATquecaracterizael SHU. Eneste documentoconsideramosSHUatípico(SHUa)como elsubtipode SHUen elquelosfenómenosdeMATsonfundamentalmenteconsecuenciadelda ˜noproducido enelendoteliodelamicrovasculaturarenalydeotrosórganospordesregulacióndela actividaddelsistemadelcomplemento.Enlosúltimosa ˜nossehanidentificadodiversas mutaciones engenesdelsistema delcomplementoasociadosa SHUa, queexplicarían aproximadamenteel60%deloscasosdeSHUa,ysehancaracterizadofuncionalmente numerosasmutacionesypolimorfismosasociadosaSHUaquehanpermitidodeterminar quelapatologíaseproducecomoconsecuenciadeladeficienteregulacióndelaactivación delcomplementosobrelassuperficiescelularesyquellevaalda ˜noendotelialmediadopor laactivacióndelC5ydelavíaterminaldelcomplemento.Eculizumabesunanticuerpo monoclonalhumanizadoqueinhibelaactivacióndelC5,bloqueandolageneracióndela moléculaproinflamatoriaC5aylaformacióndelcomplejodeataquedemembrana.En estudiosprospectivosenpacientesconSHUasuadministraciónhademostradola inter-rupciónrápidaysostenidadelprocesodeMAT,conunamejorasignificativadelafunción renala largoplazoyunareducción importantedelanecesidad dediálisisyelcesede laterapiaplasmática.Enfuncióndelasevidenciascientíficaspublicadasylaexperiencia clínicaacumulada,elGrupoEspa ˜noldeSHUapublicamosundocumentodeconsensocon recomendacionesparaeltratamientodelaenfermedad(Nefrología2013;33(1):27–45).Enla presenteversiónonlinedeldocumentoseactualizanloscontenidossobrelaclasificación etiológicadelasMAT,lafisiopatologíadelSHUa,sudiagnósticodiferencialysumanejo terapéutico.

©2015SociedadEspa ˜noladeNefrología.PublicadoporElsevierEspaña,S.L.U.Esteesun artículoOpenAccessbajolalicenciaCCBY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Haemolytic uraemic syndrome (HUS) is a clinical entity consisting of the triad of nonimmune microangiopathic haemolytic anaemia, thrombocytopenia, and acute renal failure.1 _{The histological lesions of HUS typically involve}

systemicthromboticmicroangiopathy(TMA),largelyresulting inimpairedintrarenalvessels.AgreaternumberofHUScases arecausedbyaShigatoxin-producing(STEC)Escherichiacoli enteric infection or verotoxin-producing (VTEC) germs, resulting inthe so-calledtypicalHUSor STEC(VTEC)-HUS. Genetic or acquired (autoantibodies) dysregulation of the alternative complement pathway leading to endothelial

nefrologia.2015;35(5):421–447

423

damage and systemic TMA phenomena occur in nearly 10% of HUS reports.2 _{This kind of HUS related to the}

complement dysregulation is known as atypical HUS (aHUS).

In 2011, Eculizumab (Soliris®_{; Alexion} Pharmaceut-icals, Connecticut, USA) was approved by the American and European regulatory agencies for the treatment of aHUS.3 _{Eculizumab is a humanised monoclonal antibody}

inhibitingC5activation and blockingthe productionofthe proinflammatoryC5aanaphylatoxin,aswellastheformation ofthe membrane-attack complex,leading to cell lysis.4 _In

prospectivestudiesconductedinaHUSpatients,Eculizumab effectivelyhalted theTMAprocessandits effects,andwas associatedwiththerapid,significant,andlong-term improve-mentofhaematological and renal functionabnormalities,5

and with improved systemic involvement and high blood pressure.

In2012,theSpanishGroupforaHUSgatheredtodevelopa consensusdocumentincluding recommendationsfor treat-ing the disease.6 _{The group has been meeting every year}

eversincetheninorder toupdateboththe understanding of the various aspects of interest related to the dis-ease (including the aetiological classificationof TMAs, the pathophysiology ofaHUS, andcompanion diagnostics) and treatment recommendations based on already published

scientificevidenceandclinicalexperience.Thecontents orig-inally published in Nephrology 2013;33(1):27–45 have been updated in the current online version of the consensus document.

Aetiological

classification

of

thrombotic

microangiopathies

The term TMA describes a histological lesion ofthe arte-rioles and capillaries resulting in thickened and swollen vessel walls, detachment of endothelialcells, widening of thesubendothelialspacecausedbythebuild-upofproteins andcelllysismaterial,andthepresenceofplateletthrombi obstructingvascularlumen(Fig.1).1_{Twoclinicalentitieswith}

differentaetiologyandpathophysiologyarecharacterisedby primaryTMAlesions:thromboticthrombocytopenicpurpura (TTP)andHUS.

IntravascularthrombosisinTTPresultsfromasevere defi-ciency in the metalloprotease activity of the A Desintegrin andMetalloproteinasewithThromboSpondintype1motif, mem-ber13(ADAMTS13),aplasmaenzymeresponsibleforsplitting theultra-largemultimersoftheVonWillebrandfactor.7_This

deficiency may be genetic or acquired via IgG circulating

Fig.1–Renalhistopathologicallesionsfromhaemolyticuraemicsyndrome.(A)Ischaemicandretractedglomeruli.(B) Mesangiolysis(C)Thrombiintheglomerularcapillaries(arrow).(D)Arteryoccludedbyplateletthrombi.Photographs courtesyofDr.R.Ortega(HistopathologydepartmentoftheHospitalUniversitarioReinaSofía,Córdoba).

antibodies blocking ADAMTS13 (particularly in patients receivingplateletantiaggregants).8

NinetypercentofHUScasesarecausedbyaSTECenteric infectionresulting fromcontaminated food(typical HUSor STEC [VTEC]-HUS).2 _{TheShiga toxin causes a directinjury}

on the vascular endothelium, triggering a number of cell andvasculareventswhichultimatelyleadtoTMA.2_Clinical

presentationusuallyinvolvesabdominalpainanddiarrhoea, togetherwithacuterenalfailurewithin4–10days.Prognosis istypicallyfavourable,withamortalityratebelow5%and80% ofpatientsachievingcompleteclinicalrecovery,although pro-gressiontoseverechronicrenalfailureisobservedovertime inupto20–30%ofpatients.9,10

aHUS is essentially diagnosed by exclusion once ADAMTS13 (TTP) deficiency or STEC infection (STEC-HUS) are ruled out. In patients with aHUS, TMA phenomena are a consequence of the dysregulation of the alternative complement pathway on the cell surface. This abnormal-ity results in uncontrolled activity on own cells following complement activation (by severaltriggering factors), lead-ing to endothelial damage, inflammation, and secondary thrombosis, with an increasingnumber of casesinvolving geneticoracquired factors.Mutationshavebeen described inoneor morecomplement proteinsinnearly60% ofover 1000 patients with reports of aHUS in the literature,11–18

althoughageneticcomponent(involvingcomplementgenes or other, including coagulation genes) and/or unspecified autoimmunitymayalsobepresentintheremainingpatients. Ofnote, anti-FactorH(FH) autoantibodieshavebeen found in 5–10% of aHUS patients.19 _{Unlike STEC-HUS, which is}

usually an isolated event, aHUS is a chronic and relaps-ing entity triggered by the uncontrolled activation of the complement system. Before Eculizumab became available, aHUShadbeenmostlyassociatedwithapoorprognosis:the mortalityratefollowingafirstepisodeofaHUSwas10–15%, and renal function remained unrecovered in up to 50% of patients.11,12,20

AtypeofaHUSresultingfromrecessivemutationsinthe DGKEgenecodingfortheDGK-␧(diacylglycerolkinase-␧) pro-teinhasrecentlybeendescribed.21 _{Thelossofthisenzyme}

activityinendothelialcells,platelets,andpodocytesleadsto endothelialcellsapoptosisandimpairedangiogenicresponse, thereby resulting in a prothrombotic and inflammatory state.22_{PatientswithDGKEmutationsexhibitvarious}

pheno-typesrangingfromaHUStomembranoproliferative glomeru-lonephritiswithhighproteinuriaandnephroticsyndrome.23

aHUS patients develop persistent high blood pressure and haematuria-proteinuria(includinginthenephroticrange)in theirfirstyearoflife.UnlikepaediatricaHUSassociatedwith complementgeneticalterations,progressiontochronicrenal diseaseamongthesepatientsisnotsudden,butdevelopsover years.21

In addition to STEC enteric infection (typical HUS), abnormalities in the regulation of complement activation, mutationsinDGKEorcoagulationgenes(aHUS),or(genetic or autoimmune)deficiency ofADAMTS13inTTP, thereare many other factors and clinical entities thatmay be asso-ciated with the development of TMA. This kind of TMA is included under the term secondary TMA. Some cases reported in children are associated with methylmalonic

aciduria24 _{or more commonly (5% of HUS reports in}

chil-dren) with neuraminidase-producing invasive Streptococcus pneumoniaeinfections(resultingintheexposureofthe crypto-antigen T in the cell surface and unleashing the TMA phenomenon),25 _{or H1N1infection.}26 _TMAhasbeen

gener-ally associatedwithviral infections(CMV, HIV,parvovirus), neoplasticprocesses,drugs(antitumoragents,includingthe vascular endothelialgrowth factor inhibitors, immunosup-pressants such as calcineurin inhibitors [cyclosporine and tacrolimus]orthemammaliantargetofrapamycininhibitors [mTOR;sirolimus,everolimus],plateletantiaggregants, antivi-rals, or oral contraceptive drugs), malignant high blood pressure,bonemarroworsolidorgantransplantation, preg-nancy and postpartum, autoimmune systemic diseases, or glomerulonephritis.27

Importantly,theforegoingcausesofTMAmaynotalways be identified in all patients, whereas some may present morethan oneaetiology,resultinginaheterogeneous pre-sentation and achallenging diagnosis. Infact, overlapping entitiesare commonandup to25% ofpatientswith STEC-HUS and 86% of patients with pregnancy-associated HUS developcomplementsystemmutations,whereaHUSis actu-allytheunderlyingdisease.28,29_{Mutationsinthecomplement}

systemhavealsobeenreportedinpost-transplantHUS asso-ciated with the use of calcineurin inhibitors and in HUS relatedtoautoimmunediseasesin27%and33%ofpatients, respectively.12_{Furthermore,severalcasesofsecondaryTMA}

have been reportedtodate withsuccessfultreatment out-comeswithEculizumab(TMAassociatedwithdrugs,30_solid

organs31_orbonemarrow32_{transplantation,pregnancy}33_and

systemic erythematouslupus34_{).Thefactthat complement}

blockade (by Eculizumab) is associated with a favourable clinical responseand the reversibility ofTMAsuggests the potentialandimportantroleofnon-geneticcomplement dys-regulation in many cases of secondary TMA, predisposing patients to its development. On this basis, Fig. 2 sum-marisestheproposedaetiologicalclassificationofTMAsand illustrates the potentialoverlapping betweenthese clinical entities. The classification of TMAs should be understood as a current topic of interest and major debate is tak-ing place among the medical community as a result of the continuousprogressmade intheunderstanding ofthe pathophysiology of these entities.35 _{Given that the aHUS}

mediatedbycomplementdysregulationisthe mainreason fordebate,onlythisentitywillbediscussedinthefollowing sections.

Atypical

haemolytic

urinary

syndrome:

a

clinical

entity

Epidemiology

aHUSisconsideredanultra-raredisease.Dataavailableonits incidenceandprevalencearelimited,aswellasthe knowl-edgeoftheactualepidemiologyofthedisease.Theannual incidence of aHUS in the US has been estimated to beof ∼1–2cases/millioninhabitants.36_{Arecentmulticentrestudy}

inEuropereportedanincidenceof0.11cases/million inhabi-tants.AccordingtotheEuropeanMedicinesAgency(EMA),the

nefrologia.2015;35(5):421–447

425

TTP

Secondary

TMAs

aHUS

(associated with

complement)

Shiga toxin-producing Escherichia coli - Strain 0157:H7 and other strains - Shigella disenteriae type I

- Streptococcus pneumoniae (neuraminidase) Complement abnormalities

- Mutations in FH, MCP, FI THBD, FB and C3

- Polymorphism risk in FH and MCP - Anti-FH antibodies

STEC- HUS

and STEC-like

Pregnancy

- Pre-eclampsia and eclampsia - Anti-tumour - HELLP syndrome - Mitomycin C. - Postpartum TMA Gemcitabine, Cisplatin Systemic diseases - VEGF and tyrosine kinase - SLE inhibitors (sunitinib,

- Vasculitis imatinib and dasatinib) - Scleroderma - Everolimus - Anti-phospholipid syndrome - Radiation Glomerulonephritis - Others - C3 Glomerulopathies - Quinine - Glomerulopathies associated with - Interferon

monoclonal gammapathies - Ticlopidine and clopidogrel of uncertain significance - Valaciclovir

- IgA nephropathy - Oral contraceptives Malignant arterial hypertension Bone marrow transplant Infections Solid organ transplant - HIV - Drugs

- HCV Immunosuppressants - H1N1 (influenza A) - Humoral rejection - Others Neoplasms Drugs/treatments - Immunosuppressants - Calcineurin inhibitors (cyclosporine, tacrolimus) - mTOR inhibitors (sirolimus, everolimus)

- Viral infections (CMV and BK virus)

Other causes of TMA - Methylmalonic aciduria - Intestinal lymphangiectasis

Fig.2–Classificationoftheaetiologiesofthromboticmicroangiopathies.ADAMTS13:ADisintegrinAndMetalloproteinase withaThromboSpondintype1motif,member13;aHUS:atypicalhaemolyticuraemicsyndrome;CMV:cytomegalovirus;FB: complementfactorB;FH:complementfactorH;FI:complementfactorI;HCV:hepatitisCvirus;HELLP:Hemolysis,Elevated Liverenzymes,LowPlateletcount;HIV:humanimmunodeficiencyvirus;HUS:haemolyticuraemicsyndrome;MCP: membranecofactorprotein;mTOR:mammaliantargetofRapamycin;SEL:systemicerythematouslupus;STEC:Shiga toxin-producingEscherichiacoli;THBD:thrombomodulin;TMA:thromboticmicroangiopathy;TTP:thrombocytopenic thromboticpurpura;VEGF:vascularendothelialgrowthfactor.

prevalencemay beof∼3.3patients/millioninhabitants/year amongpatientsbelowtheageof18,withlowerratesamong adults.

ChildrenandadultsarepredominantlyaffectedbyaHUS, althoughitmaydevelopatanytimeinlife.11,12_{Theonsetofthe}

diseaseusuallyoccursbeforetheageof18(60%vs.40%)and sexcharacteristics arewell-balanced (womenare primarily affectedwhenthediseaseisdevelopedinadulthood).11,13

Clinicalpresentation

Clinicalonsetisoftenabrupt,although20%ofpatientsmay developitprogressively(inweeksormonths),accompaniedby subclinicalanaemia,fluctuatingthrombocytopenia,and pre-servedrenal function.11 _{Theclinical picture comprises the}

triadofnonimmunemicroangiopathichaemolyticanaemia, thrombocytopenia, and acute renal failure.1 _{High levels}

of lactate dehydrogenase (LDH), undetectable haptoglobin and schistocytes confirm the presence of intravascular hemolysis20_{associatedwithhaematuria,proteinuria,and/or}

acuterenalfailure(withorwithoutoligoanuria).Highblood pressureresultingfromvolumeoverloadorvascularlesionis common.1_{Insomepatients,thesinglemanifestationofTMA}

maybeproteinuriawithhighbloodpressureandprogressive renalfailurewithouthaematologicalabnormalities.

Even though aHUS lesions are predominantly observed in renal vessels, the diffuse and systemic nature of the TMAphenomenonleadstotheinvolvementofthe microvas-culature of other organs (including, but not limited to the brain,heart,intestine, pancreas, and lungs),1 _therefore

accounting for common extrarenal symptoms.11,12

Neuro-logical symptomsare the mostcommon (48%),37 _including

irritability, somnolence, confusion,convulsions, encephalo-pathy,stroke,hemiparesis,visualabnormalities,hemiplegia, and coma.1,12,37,38 _{Myocardialinfarctionhasbeendescribed}

inupto3%ofaHUSpatientsinrelationtosuddendeath.12,39

Myocardiopathy, heart failure, and peripheral ischaemic vasculopathy have also been reported,19,37,40,41 _{as well as}

diarrhoea (30%) and other digestive symptoms (including, but not limited to colitis, nausea, vomit, abdominal pain,

hepatitis,cholestasis,andpancreatitis).12,19,38,42_Skin

involve-mentincludingulcerlesionsinlowerlimbshasrecentlybeen reportedinaHUSpatients.43_{Heterogeneityofsymptomshas}

posedachallengeforcompaniondiagnosticsofothercauses ofTMA.

Pathophysiology

The complement system, consisting of several circulating plasmaandmembrane-associatedproteins,ispartofinnate immunity and is vital for fighting infections, processing immune complexes, antibody response, and the elimina-tionofapoptotic residues.Activationbyanyoftheexisting pathways(classical,lentin,andalternative)leadstothe for-mationofmultiproteincomplexeswithC3-convertaseactivity splitting the C3 protein and resulting in C3b (Fig. 3). The covalentbindingofthismoleculetothesurfacesactivating thecomplementfavoursphagocytosisbypolymorphonuclear leukocytesandmacrophages,thereforeresultinginactivated C5,directingtheattackcomplextothemembraneand caus-ingcelllysis.Inaddition,theresultingC3bleadstoarapidly enhancedcomplementactivationbypromotingtheformation of further C3-convertases, since it is one of the compo-nentsofC3-convertaseofthealternativepathway.44_Inorder

toavoidtotaluptakebycomplement activation,aswell as damageto selftissues (C3bbinds indiscriminately to both pathogens and self cells), a number of process-regulating proteins,suchasFH,themembranecofactorprotein(MCP), andcomplement factorI(FI)dissociate C3-convertasesand resultinC3bdegradation.C3blevels,therefore,remainlow undernormalconditionsandtheybuildup following com-plement activation only in the structures related to this activity.

Several studies have shown that around 60% of aHUS patientsarecarriersofmutationsincomplement-regulating genes(CFH,MCP,CFI,thrombomodulin[THBD],orinthe com-ponentsofC3-convertase,factorB[FB],andC3).45–54_Allthese

mutationscausethedysregulationofthealternativepathway (Table 1). FH acts in plasma by controlling complement homeostasisandincellsurfacesbypreventingdamagetoself components. Mutationsin theC-terminal regionofFH are characteristicofaHUS.BecausethealteredFHregion medi-atescomplement activationincell surfaces,cell protection againstaccidentaldamageresultingfromcomplement acti-vationisdecreasedbythesemutations,withnoinvolvement ofcomplementregulationinplasma.55_{Thefunctionalassay}

ofaHUS-associated mutationsfound in other complement genes,includingMCP,CFI,CFBorC3,hasalsoconfirmedthat all ofthemresult inadefective protectionofcell surfaces and thislossofcomplement regulationmay beduetothe decreasedactivityofregulatingproteinsortotheabnormally high activity ofC3-convertases. Thus,while theregulatory activityoftheseproteinsisimpairedbymutationsinFH,MCP, andFI,mutationsinFBorC3resultinfurtheractivationofthe C3-convertase.

Around5–10%ofaHUSpatientsdevelopanti-FHantibodies targetedtotheC-terminalregion,withsimilareffectstothose observedinFHmutations.56,57_{Theirroleinthepathogenesis}

of aHUS has not been fully established, but seems to be

Table1–Riskfactorsinatypicaluraemichaemolytic

syndromea Mutations Lossoffunction CFH(∼13%) MCP(∼11%) CFI(∼10%) THBD(∼4%) Gainoffunction C3(∼4%) CFB(∼3%) Polymorphisms Increasingrisk CFH:c.-332C>T;c.2016A>G(p.Gln672Gln);c.2808G>T (p.Glu936Asp)

MCP:c.-652A>G;c.-366A>G;c.989-78G>A;*897T>C

Providingprotection CFH:c.184G>A(p.Val62Ile) Autoantibodies Anti-FH(∼5%) Environmentalfactors Infections Immunosuppressants Oralcontraceptives Anti-cancerdrugs

Anti-FH:anti-complementfactorHantibody;CFB:complement

fac-torBgene;CFH:complementfactorHgene;CFI:complementfactor

Igene;MCP:membranecofactorprotein.Gene;THBD:

thrombomo-dulingene.

a _{“Multiplehits”theory.aHUSisacomplexdiseasenormally}

involv-ingvariousrisk,genetic,andenvironmentalfactors.Patientsare

commonlycarriersofmorethanonemutationincomplement

genesorcombinedmutationswithriskpolymorphisms.

Envi-ronmentalfactorsarealsonecessarytohelprevealthegenetic

disposition from mutations or polymorphisms. Concomitant

mutationswithriskpolymorphisms,autoantibodies,or

trigger-ingenvironmentalfactorsaccountfortheincompletepenetrance

ofaHUS,aswellasforthedifferencesinitspresentationand

progressionamongcarriersofcomplementgenemutations.

associatedwithdiseaseonsetorrecurrence.Giventhat anti-bodytitresmaydecreaseovertime,theyshouldbescreened earlyinthecourseofaHUS.Anti-FHantibodiesareassociated withcomplementfactorH-relatedprotein1deficiency(FHR1) inpatientswithaHUS.58

PenetranceofaHUSincarriersofmutationsinsomegenes isaround 50%,withonlyafewcarriersfrom familieswith identifiedmutationscommonlydevelopingaHUSand show-ing a variable clinical presentation. Clinical heterogeneity, whichresultsfromthe existenceofadditional(genetic and environmental)riskfactorsmediatingthedevelopmentand theoutcomeofthedisease,islargelyobservedamong unre-lated carriers of this mutation. Screening for complement mutationsinaHUSpatientsandconductingcase-control stud-ies basedon geneticpolymorphismsincandidate genesor geneticmarkersinthehumangenomehasallowedforthe identificationofsomevariants(polymorphisms)inCFHand MCPgenesmodulatingthepenetranceandseverityofthe dis-ease(Table1).49,59,60

Haplotypes CFH-H3 and MCPggaac are the most rele-vantpolymorphismsassociatedwiththeriskofaHUS.Both

nefrologia.2015;35(5):421–447

427

Fig.3–Complementdysregulationinatypicalhaemolyticuraemicsyndrome.Complementactivationbyanyofthe3 pathways(detectionofforeignantigens,alternativepathway;ofantibodies,classical;ormannanpolysaccharides,lectin) leadstothebuild-upoflargequantitiesofC3bontheactivatorcellmembrane,causingopsonisationandC5activation (terminalorlyticpathway),resultingintheformationofthemembraneattackcomplexandcelllysis.Complement activationresultsininflammationandleucocyterecruitment.ThekeyprocessincomplementactivationisC3bformation, whichdependsonunstableenzymaticcomplexes–C3-convertases–catalysingtheruptureofC3tocreateC3b.Inturn,C3b hastheabilitytoformfurtherC3-convertaseofthealternativepathway(C3bBb),thusenhancingtheinitialactivation.The mediationofC3Bproductionistwo-fold:dissociationofC3-convertasesandproteolyticinactivationofC3bandC4b.Several regulatoryproteinsinplasmaandthecellmembranecarryoutthisregulatoryactivities,including,factorH,MCPandfactor I,whichplayanessentialroleinthedissociationofC3-convertaseofthealternativepathway(C3bBb)andtheproteolytic degradationofC3b.MutationsintheseproteinsfoundinpatientswithaHUSinterferewiththisregulatoryactivityofthe alternativepathwayactivation.SomepatientswithaHUSarecarriersofmutationsinproteinsC3andfactorBorganising C3-convertase.Thesemutationsareparticular,astheyincreasetheactivityofmutatedproteins(gain-of-function mutations),resultinginincreasedcomplementactivationandexceedingthecapacityofregulatoryproteins.

haplotypesincludesingle-nucleotidepolymorphisms(SNP)in theCFHandMCPgene-promoterregion,downregulatingFH andMCP.Thepresenceofbothpolymorphismsin homozygo-sismayprovidearationaleforaHUSdispositioninpatients withnomutationsinanyofthegenesassociatedwithaHUS. ArecentcollaborativestudybytheEuropeanWorkingParty on Complement Genetics in Renal Diseases including 795 patientswithaHUShasshownthat3%ofthesepatientswere carriersofcombinedmutationsinmorethanonegene. Addi-tionally,this largestudy hasproved that concomitant risk haplotypesCFH-H3 and MCPggaac also leadto significantly increaseddiseasepenetranceincarriersofcombined muta-tions,stressingtheideathatgenotypingoftheserisk polymor-phismshelpspredicttheriskofaHUSinmutationcarriers.61

Alongwiththepreviousgeneticalterations,anumberof triggeringenvironmental factorsare also implicatedinthe onsetofaHUS.Theabovemutationsarepredisposingfactors ofthedisease,preventingadequatecomplementregulationin cellsurfaceswhenthesystembecomesactivatedin microves-sels. aHUS is triggered by infectious events in 50–80% of

patients,11,12,40_{particularlythoseinvolving theupper}

respi-ratorytract(influenzaH1N1virus).Diarrhoeacausedby gas-troenteritismayprecedeaHUSinupto30%ofcases(including diarrhoea by STEC11,12,19_).12 _{Pregnancy, particularly during}

post-partum,isacommonpredisposingfactorofaHUSamong women,12,29_{togetherwiththeuseoforalanovulatoryagents.}

Mutationsinthegenecodingforthrombomodulin(THBD), ananticoagulantproteinactingasthrombincofactorandalso regulatingtheFI-mediatedC3binactivation,havebeen associ-atedwithaHUS.62_{Basedoncomplementdysregulationtypical}

ofpatientswithaHUS,thefunctionalanalysisofTHBD muta-tionsassociatedwithaHUShasshownthatthrombomodulin mutationsimpairthecomplementregulatoryactivity.62

Nev-ertheless,theimpairmentoftheanticoagulationactivityby thrombodulinmutationsassociatedwithaHUSand the rel-evance ofthese abnormalities inaHUS remains unknown. Inthisregard,arecentstudyconductedin36patientswith aHUS hasassessedthepresenceofmutationsinthegenes of the complement system and coagulation through mas-siveDNAsequencing,detectingmutationsingenesfromboth

Table2–Clinicaloutcomeofpatientswithatypicalhaemolyticuraemicsyndromebasedoncomplementabnormalities (priortoEculizumab).

Gene RiskofdeathorESRFin

thefirstepisodeor

withinthenextyear

Riskofrelapse RiskofdeathorESRFat

3–5years

Riskofrelapsefollowing

renaltransplant CFH 50–70% 50% 75% 75–90% CFI 50% 10–30% 50–60% 45–80% MCP 0–6% 70–90% 6–38%a _<20% C3 60% 50% 75% 40–70% CFB 50% 3/3withoutESRF 75% 100% THBD 50% 30% 54%a _1patient

Anti-FH 30–40% 40–60% 35–60%a _{Higherwithincreased}

antibodytitres

Anti-FH:anti-complementfactorHantibodies;CFB:complementfactorBgene;CFH:complementfactorHgene;CFI:complementfactorIgene;

ESRF:end-stagerenalfailure;MCP:membranecofactorproteingene;THBD:thrombomodulingene.

a _DataonESRF.

AdaptedfromLoiratandFremeaux-Bacchi.1

systems.63_{Thegeneinthecoagulationsystemwiththelargest}

number ofmutations was plasminogen (PLG), a cymogene whichplaysanimportantroleinfibrinolysisfollowing con-versiontoplasmin.Eventhoughthesedatasuggestthat coag-ulationgenesmayaddtoaHUSdisposition(particularlyPLG), furtherstudiesarerequiredtoconfirmtheseobservations.

Thesearch ofnewgenesassociatedwithaHUShasalso beenaddressedbyLemaireetal.21_throughexome

sequenc-ing.Theauthorshaveidentifiedhomozygoticmutationsinthe DGKEgenecodingfortheDGK-␧proteinin13patientswith aHUSfrom9families.Thesepatientshadaveryearlyonset ofaHUS,generallywithintheirfirstyearoflife,followedby multiplerecurrencesandcommonprogressiontoend-stage renalfailureintheseconddecadeoflife.21_Deficiencyof

DGK-␧inendothelialcellshasbeenrecentlyshowntoinducethe expressionofICAM-1andtissuefactorbymeansofincreased p36-MAPK-mediatedsignalling,leadingtoapoptosis,altering theangiogenicresponse,anddeterminingaproinflammatory andprothromboticphenotype.Yet,theabsenceofDGK-␧is notdetrimentaltocomplementactivationincellsurfaces.21,22

The absence of DGK-␧ in podocytes and endothelial cells mayprobablyimpairthediaphragmofglomerularfiltration, whichwould accountformassiveproteinuriaand the sus-ceptibilitytoglomerularconditionsamongthesepatients,21,23

althoughthereasonwhythesepatientstendtodevelop sev-eralglomerularconditionsremainsuncertain.Finally,despite theroleofthecomplementinthedevelopmentofrenal dis-ease amongcarriersofDGKE, mutationshad beeninitially ruledout,21_{patientswithDGKEmutationsadditionally}

asso-ciatedwithothergenespreviouslyrelatedtoaHUS,including THBDandC3,64_{havebeenrecentlyidentified,thussuggesting}

thatcomplementdysregulationmayplayaroleinthe modu-lationofdiseaseonsetandoutcomeatleastinsomecarriers ofDGKEmutations.

Prognosis

TheavailabilityofEculizumabhassignificantlyrevolutionised the prognosis of patients living with aHUS, a very severe

disease in mostcasesin spiteofintensive treatmentwith plasmatherapy(PT;Table2).FollowingafirstepisodeofaHUS, overall mortalitywas higherthan 10% and morethan half of the patients required dialysis and/or developed a more permanent renaldamageinthenext12months.11,12,20_The

clinicaloutcomechangesrelatedlydependingonthepatient’s mutation.Inthisrespect,outcomeseemedtobeparticularly poorinpatients withFHandC3mutations,withmortality and end-stage chronicrenal failure (ESCRF)rates over 50% withinoneyearfromthefirstepisodeofaHUS.Furthermore, halfofthesepatientsrelapsed.MutationsinFI,FB,andTHBD werealsoassociatedwithhighratesofmortalityandESCRF atoneyear (50%),withrelapses occurringinnearlyonein 3 patients overcoming the first episode of aHUS. On the other side, less than 10% of patients withMCP mutations died or progressed to ESCRF, although the risk of relapse among these patients was higher and up to 90% of them developed new episodes of aHUS. Between 50 and 75% of patients withmutationsin FH,CI,C3, FB orTHBD died or developedESCRFwithin3–5 yearsfrom thefirstepisodeof aHUS.1

Recurrenceofatypicalhaemolyticuraemicsyndrome followingrenaltransplantation

The outcome ofrenal transplantation (RT) amongpatients withESCRFduetoaHUShasbeenhistoricallylimitedbythe increasedpercentageofrecurrencesofpost-transplant dis-ease(∼50%;graftlossrate:80–90%65,66_{),althoughitchanges}

significantlybasedonthetypeofalteration.Inaseriesof57 patientswithaHUSreceivingRT,5-yearrecurrence-freegraft survivalwas significantlylower inpatientswithmutations in the genes coding for complement proteins compared to patients in whom only polymorphisms but no genetic abnormalities were found.67 _{Yet, itshould bestressed that}

theriskofrecurrenceofaHUSfollowingRTinpatientswith nogeneticabnormalitiesisalsodeemedhigh.68_Mutations

in FH are associated with a higher risk of recurrence or graft lossfollowingRT(75–90%;specificallythoserelatedto

n e f r o l o g i a. 2 0 1 5; 3 5(5) :421–447

429

Table3–Diagnostictestsandproceduresrecommendedforpatientswiththromboticmicroangiopathy.

Generaldiagnostictests

•Completemedicalrecords,includingdrugs,datafromsystemicdiseases,personalandfamilyhistory

•Completephysicalexamination,includingafundoscopicexam

•Generalroutinebloodandurinetests

•Haptoglobinlevels

•Serumcomplementlevels

•Peripheralbloodsmear

•Serologyforsystemicdiseases(ANA,anti-ADN,ANCA,antic-Scl-70,anticentromere)

•Anti-cardiolipinantibodiesandlupusanticoagulant

•SerologyforHIV,HCV,HBV,CMVandH1N1

•Completeclottingtest,withfibrinogen,fibrinogendegradationproductsanddimerD

•InvestigationsfortypicalHUS-causingbacterialinfectionsandShigatoxintest(ifclinicallysuspected)

Specificdiagnostictests

•STECinfection •Faecalsampleincaseofdiarrhoeaorrectalculture:STECcultures(MacConkeyforE.coliO157:H7);PCRforStxgenesO157:H7andother

serotypes,andotherviruscharacteristics;ELISAand/orVerocelltissuecultureassayforStxserum:anti-LPSantibodiesforprevalentserotypes

•Pneumococcalinfection •Bacterialculture(generally)ofsterilebodyfluids;DAT(Coombstest),viraltest(respiratory),chestx-ray(pleuraleffusionasacharacteristicin

mostcases),cytochemistry,andCSFcultureincasestopneumococcalmeningitis

•Alteredregulationofthecomplement •C3,C4(plasma/serum),AH50

•FH,FI,FB(plasma/serum)

•Anti-FHautoantibodies

•ExpressionofsuperficialMCPinleukocytes(poly-ormononuclearleukocytesusingaFACStest)

•MutationanalysisinFH,FI,MCP,C3,FB±THBD

•ADAMTS13deficiency(acquiredorhereditary) •PlasmaactivityofADAMTS13ordose(ELISA)±inhibitor

•Cobalaminmetabolism:methylmalonicaciduria •Aminoacidchromatographyinplasma/urine(hyperhomocysteinemia,hypomethioninemia;homocystinuria);organicacidchromatographyin

urine(methylmalonicaciduria)

•MutationanalysisforthegeneMMACHC

ADAMTS13:ADisintegrinAndMetalloproteinasewithaThromboSpondintype1motif,member13;ANA:antinuclearantibody;ANCA:Autoantibodiestoneutrophilcytoplasmicantigens;CMV:

cytomegalovirus;CSF:cerebrospinalfluid;DAT:directantiglobulintest;DNA:deoxyribonucleicacid;ELISA:enzyme-linkedimmunoabsorptionassay;FACS:fluorescenceactivatedcellsorting;FB:

complementfactorB;FH:complementfactorH;FI:complementfactorI;HIV:humanimmunodeficiencyvirus;HUS:haemolyticuraemicsyndrome;MCP:membranecofactorprotein;STEC:Shiga

toxin-producingEscherichiacoli;THBD:thrombomodulin;VHB:hepatitisBvirus;VHC:hepatitisCvirus.

abnormalities interminal 3′ _{and gene conversion between} CFHandCFHR1,resultinginthehybridgeneCFH/CFHR1[both abnormalities impair the functionality of the C-terminal domain of FH]), posing a high risk with mutations in C3 andFIaswell(40–80%;Table2).12,42,48,65,67,69–71_Todate,very

few transplants have been performed in patients with FB mutations,though recurrence ofaHUS and graft losswere reported inall cases.49,72 _{In general,plasma factors ofthe}

complement involvedinaHUSare synthesisedinthe liver, and so patients with mutations in the complement genes codingforthesefactorsremainpronetoaHUSfollowingRT, asdysfunctional factors are stillbeing produced.MCP is a transmembraneproteinthatishighlyexpressedinthekidney and,asaresult,thisdefectcanbecorrectedbyRTby deliver-ingunchangedMCPintothegraft.Over80%ofpatientswith MCPmutationsdevelopnorecurrenceofaHUSfollowingRT, withalong-termsurvivalratecomparabletothatofpatients receivingtransplantsforotherreasons.40,65,66_Theriskof

post-transplantrecurrenceinpatientswithTHBD62_mutationsor

anti-FHantibodiesisnotwell-established,althoughitseems toberelatedtohighandpersistenttitresofantibodiesinthe latter.19,73

Diagnosis

of

atypical

haemolytic

uraemic

syndrome

In light ofthe rapid evolution and the severity ofTMA, a differential diagnosis should be immediately established fromthesyndromeperspective,allowingforsupportive mea-sures tobetaken within24–48hfrom patient’s admission. ConsiderationsforanaetiologicaldiagnosisofTMAwill sub-sequentlybemade.Table3summarisesthemajorprocedures anddiagnostictestsrecommendedforthediagnosisofTMA, including specific tests for companion diagnostics of the variousaetiologiesofTMA.

In patients with TMA, tests results include thrombo-cytopenia (platelet count <150,000/mm3 _{or decrease >25%} frombaseline)20_{and microangiopathichaemolyticanaemia}

(haemoglobin<10mg/dlwithanegativedirectCoombs test [thoughsomepatientswithpneumococcalorH1N1-related HUSmayshowpositivedirectCoombstest],25_elevatedLDH,

decreasedhaptoglobin,reticulocytosis,andschistocytes).20,62

Inaretrospectiveseriesof50patientswithhistologicalTMA, 44% had a normal platelet count.74 _{Consequently,}

diagno-sis of TMA should be considered in patients with renal failure and elevated LDH, but without thrombocytopenia. For schistocytes, even though they can be found in most patientswithrenaldisease,preeclampsiaormechanicvalves, TMA can be diagnosed with a schistocytecount >1% pro-vided thatthereare no other known causes.75 _{In contrast,}

the absence of schistocytes does not rule out a diagnosis ofTMA.

Highlevelsofserumcreatinine,lowglomerularfiltration (GF)orthepresenceofproteinuriaand/orhaematuria11,20,69

areindicativeofrenalfailure.Arenalbiopsymayberequired foradultpatientsfollowingacuterenalfailuretodetermine the aetiology,rule out other processes, and assess progno-sis,although the indicationand timefor biopsiesmust be examinedindividuallyinpatientswithsuspectedTMAdue

Table4–Differentialdiagnosisbetweendisseminated

intravascularcoagulationandthrombotic

microangiopathy.

DIC TMA

Plateletcount ↓ ↓

Fibrinogen ↓ Normal

Fibrinogendegradationproducts ↑ Normal

DimerD ↑ Normal

Antithrombin ↓ Normal

Schistocytes Present Present

Haptoblogin Normal ↓

Coagulationtimes Long Normal

Bloodpressure ↓ ↑

DIC: disseminated intravascular coagulation; TMA: thrombotic

microangiopathy.

totheriskofbleeding.Inthissense,diagnostic renal biop-siesarenotrecommendedinpatientsconclusivelydiagnosed withaHUS(positivefamilyhistory,recurrence,etc.).Among paediatricpatients,thediagnosisisessentiallymadeonthe basisofclinicalpresentation,thoughrenalbiopsymay occa-sionallyberequired(especiallyincasesofsecondaryTMAor RT). Patients withclinical suspicion ofTMAshould always beexamined byanephrologistinlightofthe urgent treat-mentstrategyrequiredtoensureminimumirreversiblerenal damage.

Disseminated intravascular coagulation (DIC) is a syn-dromethatmaybeassociatedwithseveralmajorlaboratory and clinical findings related to TMA. DIC is characterised by a systemic activation ofcoagulation, secondary to sev-eral clinicalconditions(sepsis,traumaorcertain tumours), leading to thrombosis and bleeding, commonly involving renalfunction.76_{Keytestcriteriabasedoncoagulationtests}

for differential diagnosis of DIC and TMA are listed in Table4.

Acompleteanddetailedclinicalhistoryshouldbemade forTMApatients,includingpersonalandfamilyhistory, trigg-ering factors (drugs, infections), and a thorough physical examination. As opposed to previous considerations made severalyearsago,signsandsymptomsofthedifferenttypes ofTMAarecurrentlythoughttobenonspecificandavoidthe companiondiagnosticsbetweenthesetwoentities.1_The

dif-ferentiationbetweenHUSandTTPwasclassicallybasedon clinical criteria, with HUS and TTP being diagnosed when renal involvementand neurological involvementwere pre-dominant, respectively. However,50% ofpatients withTTP developrenalfailureand50%ofpatientswithaHUSdevelop neurologicalabnormalities.37,77

Clinicalfeaturesdonotallowforadifferentiationbetween STEC-HUS andaHUSaswell,giventhatupto30%ofaHUS cases are developed following gastroenteritis12 _{or patients}

develop diarrhoea42 _{(a typicalsymptom ofSTEC-HUS). On}

the other hand, platelet count and the severity of renal involvementcanactuallyguidecompaniondiagnostics. Over-all,TTPpresentswithseverethrombocytopenia(<20,000/mm3 in 73% of patients with acquired TTP)78 _{and moderate}

nefrologia.2015;35(5):421–447

431

Thrombocytopenia

±

Microangiopathic haemolysis

Plus one or more than one of the following

Evaluate ADAMTS13 and Shiga toxin /STEC** test

TTP

<5-10% ADAMTS13 activity

>5-10% ADAMTS13 activity

Shiga toxin /STEC positive

aHUS

Secondary TMA

STEC-HUS***

Neurological symptoms

Confusion Seizures

Renal involvement

Elevated creatinine

Decrease in estimated glomerular filtration rate Urinary changes

Gastrointestinal involvement

Fig.4–Algorithmforthedifferentialdiagnosisofprimarythromboticmicroangiopathy.ADAMTS13:ADisintegrinAnd MetalloproteinasewithaThromboSpondintype1motif,member13;aHUS:atypicalhaemolyticuraemicsyndrome;HUS: haemolyticuraemicsyndrome;LDH:lactatedehydrogenase;STEC:ShigatoxinproducingEscherichiacoli;TTP:thrombotic thrombocytopenicpurpura.*NegativedirectCoombstest.**TheShigatoxintest/STECisindicatedwhenthepatienthasa historyofdigestiveinvolvementorgastrointestinalsymptoms.***STECinfectioncanrarelytriggertheunderlyingdisease activityinsomepatientswithaHUS.

moderate thrombocytopenia (50–100,000/mm3_{) and severe} renalinvolvement.Thisrulecanbedeemedaguide,but deter-minationofADAMTS13activityandtheShigatoxintestare essentialforanaccuratedifferentialdiagnosisbetweenTTP, STEC-HUS,andaHUS(Fig.4).ThediagnosisofSTEC-HUScan beconfirmedbythepresenceoftheShigatoxinorapositive cultureofSTECinpatientswithTMA,28_{whiletheactivityof}

ADAMTS13inplasmashouldbe<5–10%inordertoconfirm thediagnosisofTTP.79,80_{Thediagnosisoftheremainingcases}

shouldbedirectedtowardsaHUS,79_{andsoadditionaltestsare}

requiredtoruleoutsecondaryTMAs.Testsamplesshouldbe collectedpriortoPT.

Treatment

options

for

atypical

haemolytic

uraemic

syndrome

Treatment for aHUS should involve two different strate-gies:on oneside,supportivetreatmentmeasuresaimedat managing the consequences of aHUS (acute renal failure,

highbloodpressure,anaemia,thrombocytopenia,etc.),anda targetedtherapytohaltandrevertTMA.Specificoptionsfor themanagementofaHUSwillbereviewedinthissection. Plasmatherapy

PT can be delivered as plasma infusion (PI) and plasma exchange (PE). In PI, patients are given virus-inactivated, non-nativefreshfrozenplasma(FFP),addingfunctional com-plement regulators.81 _{In PE, a patient’s plasma is replaced}

with FFP, which not only results in the administration of high doses ofcomplement-regulating proteins, but also in theeliminationofdysfunctionalendogenoussoluble comple-mentinhibitors,minimisingtheriskofvolumeoverload.In addition,anti-FHantibodiesarealsoclearedinPE,together withpotential inflammatory/thrombogenicfactors involved in endothelial damage and platelet hyperaggregation. The treatmentofchoicerecommendedforepisodesofaHUS tra-ditionallyconsistedofearlyandintensivePEathighvolumes and ofvariable frequencybasedondisease activity.PIsare

Table5–Prognosisofpatientswithatypicalhaemolytic

uraemicsyndrometreatedwithplasmainfusionor

plasmaexchange. Remission Deathor end-stagerenal failure CFH 63%(complete:5%; partial:58%) 37% CFI 25%(complete: 12,5%;partial:12,5%) 75% C3 57%(complete:43%; partial:14%) 43% THBD 88%(complete:62%; partial:25%) 13%

Anti-FHantibodies 75%(complete:25%;

partial:50%) NA MCP 97%oftreated patients(complete: 90%;partial:7%)y 100%ofnon-treated patients NA

Anti-FH:anti-complementfactorHantibodies;CFH:complement

factorH gene; CFI:complement factorI gene; MCP:membrane

cofactor protein gene; NA: not available; complete remission:

haematologicalandrenal functionnormalisation;partial

remis-sion: haematological normalisation and renal sequels; THBD:

thrombomodulingene.

AdaptedfromNorisetal.12

usuallyineffective exceptin afew patients with complete deficiencyofFH82_{(circulatinglevelsofcomplementproteins}

are normalinmostpatients). Overall,PTisnotconsidered effectiveinpatientswithisolatedMCPmutations,asthisis anon-circulatingproteinattachedtothecellmembrane,with virtuallyallpatientsrelapsingfollowinganepisodeofaHUS irrespectiveoftheuseofPT.12

Even though no prospective clinical trials are available, PT has empirically been the treatment of choice in aHUS foryears asmortalityin patientswith TTP-HUSdecreased over the last 3 decades. Table 5 summarises the results ofthe largest international registry ofPT in patients with aHUS(InternationalRegistryofRecurrentandFamilialHUS/TTP), including273patients diagnosedbetween1996and 2007.12

CompletehaematologicalandrenalrecoveryrateswithPTin thisregistryaregenerallybelow50%(exceptforpatientswith mutationsinTHBDandMCP),and particularlylowratesin patientswithmutationsinFHandFI(5y12.5%).12_Mortality

and/oroutcomeofESRFaregenerallyhighin3outof4patients withFImutations.SomepapersprovethatearlyintensivePEis essentialtopreventpatientsfromdevelopingaHUS,and main-tenancecanpreventdiseaserecurrenceandESRF,11,81_though

themosteffectivemanagementstrategyisstillnotknown, noristhelong-termimpactonrenalfunction.

Concomitant immunosuppressionand PT may improve outcomes in patients with anti-FH antibodies.19,83,84 _High

antibodytitresarecorrelatedwithahigherriskofrelapseand renalsequelsinthesecases.19_{Eventhoughfurthertrialsare}

requiredtoconclude on howanti-FH antibodies are devel-opedbypatientswithaHUS,thefactthatthevastmajority

of these patients have complete deficiency of FHR1 leads totheideathattheseantibodiesare reallytargetedagainst the FHR1 protein, and that the anti-FH activity is a cross reaction resulting from the remarkable homology existing betweenthese2proteins.Thispossibilityisawarningofthe potentialanti-FHR1/anti-FHsensitisationinhomozygotesfor CFHR3-CFHR1 deletionbythe exposuretoexogenousFHR1, discouragingtheuseofPIintheseindividuals.

ThepotentialcomplicationsofPIare anaphylactic reac-tionstoFFP,hypervolemia,highbloodpressure,heartfailure, or hyperproteinaemia. The main complications of PE are obstructedvenousaccess(6%),lowbloodpressure(5%),and allergy (4%)85_{, with a higher frequency among paediatric}

patients.85_{Astudyconductedin71paediatricpatientswith}

aHUS(59treatedwithPE)showedthat80%ofchildrenhad some renal sequels within one month of follow-up, 17% weredialysis-dependant,and31%developedcatheter-related complications.86

Eculizumab

Eculizumab isahumanised monoclonalIgG2/4kappa anti-body that binds to the C5 complement protein with high affinity, blocking the excision into C5a and C5b, and pre-ventingtheformationoftheC5b-9complexoftheterminal complement (membraneattackcomplex)(Fig.3).4 _InaHUS,

the dysregulation of the alternative complement pathway leadstotheuncontrolledactivationofC5,causingadamage toselfstructuresviatheformationofthemembraneattack complex.Thisprocessisrapidlyandsustainablyreducedas aresultoftheblockadeoftheterminalcomplementpathway byEculizumab.AlargenumberofpatientswithaHUShave shownagoodclinicalresponsetothedrug(Table6).

TheefficacyandsafetyofEculizumabinaHUSwere ini-tiallyassessedintwophaseII,prospective,multicentretrials, including 37 patients older than 12 years of age and with primary or recurrent disease following RT, who received Eculizumab for 26 weeks,followed by long-termextension periods.5 _{Seventeen patients with aHUS (mean time from}

diagnosis:9.7months)withevidenceofprogressiveTMA fol-lowing ≥4 sessions of PT the week before their inclusion (C08-002)wereenrolledinthefirststudy.Thesecond study recruited20patients(meantimefromdiagnosis:48.3months) receivingPT(1sessionevery2weeksand3sessionsaweek) wherenodecrease >25%wasreportedinplatelet countfor at least 8 weeks prior to first dosing of Eculizumab (C08-003). Geneticoranti-FH antibodymutationswere observed in 76% and 70% of patients from the first and the sec-ond study, respectively. Primary outcomes in both studies were:(a)inhibitionofcomplement-mediatedTMA(study1: increasedplateletcount;study2:TMA-freepatient≥12weeks [nodecreaseinplateletcount>25%,noPTandnodialysis]), and (b) haematologicalnormalisation (≥2 normal consecu-tivemeasurements ofplatelets and LDH,with aminimum interval of 4weeks).Secondary outcomes included change intherateofdailyinterventionsforTMA(PIorPEsessions, dialysis,orboth,perpatient/day),renalfunction,qualityof life,safetyandtolerability.Primaryoutcomesreportedat26 weeksandinextensionstudiesaredescribedinTable7.With regardtoprimaryoutcome,following26weeksoftreatment,

nefrologia.2015;35(5):421–447

433

Table6–PublishedcasesofpatientswithatypicalhaemolyticuraemicsyndromereceivingEculizumab(lastupdatedin April2014).

PatientswithaHUSinnativekidneys

Reference Mutation Responsetoplasma

therapy

Baselineserum

creatinine,␮mol/l

Patientoutcome Lastserum

creatininevalue,

␮mol/l

121,122 _{Unidentified Resistanttoplasma}

exchange 265 Remissionafter3 years 35 123 _{CFH Partiallysensitiveto} plasmaexchange 80 Remissionafter10 weeks 26

124 a _{Unidentified Resistanttoplasma}

exchange

690 Recurrenceafter2

weeks

End-stagerenal

failure

125 a _{Unidentified Resistanttoplasma}

exchange

∼310 Recurrenceafter2

weeks

End-stagerenal

failure

126 _{CFHS1191LV1197A Resistanttoplasma}

infusion 108 Remissionafter15 months 44 127 _CFI p.A258T Resistanttoplasma exchange 610 Remissionafter7 months 230

38,128 b _{Unidentified Noplasmatherapy 600 Remissionafter6}

months 125 129 _CFH C611Y Intoleranceto plasmaexchange ∼230 Remissionafter24 months ∼100

130 _{Unidentified Resistanttoplasma}

exchange

∼325(dialysis) Remissionafter9

months

∼80

131 _{CFH Resistanttoplasma}

exchange

∼310(dialysis) Remissionafter18

months ∼75 132 _MCP c.286+1G>C Resistanttoplasma exchange Dialysis Haematological normalisation End-stagerenal failure

133 _{Unidentified Resistanttoplasma}

infusion

Continuous hemodiafiltration

Remissionafterone

year 18 134 _CFH 3355G>a; Asp1119Asn;SCR19 Resistanttoplasma exchange

Dialysis Remission>2.5years 26

43a _{Unidentified Sensitivetoplasma}

exchange

Dialysis Resolutionof

thrombocytopenia

andskinlesions

Dialysis

135 _{CFH Partiallysensitiveto}

plasmaexchange

723(dialysis) Remission Dialysis-free

136c _{Unidentified − ∼247(dialysis) Remissionafter6}

months

Dialysis-free

137 _{C3 Resistanttoplasma}

therapy

Dialysis Remissionafter2

years Dialysis 138 _CFH 3514G>T Resistanttoplasma therapy

∼222(dialysis) Remissionafterone

year 117 139 _C3 c3466G>A Resistanttoplasma exchange Dialysis Remission>11 months 115

140a _{C3 – Dialysis Remission Dialysis-free}

141c _CFB c.967A>C; p.Lys323Gln – 20 Favourableoutcome at6months,inspite ofthepersistenceof slightlyincreased plasmalevelsofLDH yC5b-9 Normallevels 142 _CFH p.Arg53Cys;c.157C.T Resistanttoplasma exchange

Normallevels Remission Normallevels

143c _CFH p.Lys1186Thr CFI p.Ile322Thr Partiallysensitiveto plasmaexchange Dialysis Remission 75 144 _CFI c.786delA Resistanttoplasma exchange Dialysis Remission 88

Table6–(Continued)

Renaltransplantpatients

PreventiveuseofEculizumab

Reference Mutation Previoustransplants

(number)

Responsetoplasma

therapy

Baselineserum

creatinine,␮mol/l

Patientoutcome Lastserum

creatininelevel,

␮mol/l

107 _{CFHW1183C No Sensitivetoplasma}

exchange

∼45 Norecurrence 44

108 _{CFHE1198stop No Noplasmatherapy Dialysis Norecurrence Normal}

109 _CFH/CFHR1 hybridgene No Sensitivetoplasma exchange Dialysis Norecurrence 80 110 _CFH/CFHR1 hybridgene No Sensitivetoplasma exchange

Dialysis Norecurrence Normal

111 _CFH/CFHR1

hybridgene

No Noplasmatherapy Dialysis Norecurrence 79

112 _{CFHc.3497C9T No Resistanttoplasma}

exchange

Dialysis Norecurrence 76

UseofEculizumabforthetreatmentofpost-transplantaHUSrecurrence

145a _{CFHY475S Yes(1) Resistanttoplasma}

exchange

132 Graftloss NS

146,147 _{C3R570Q Yes(1) Sensitivetoplasma}

exchange 320 2recurrencesin cases ofdelayed Eculizumab 230

97 _{Unspecified No Resistanttoplasma}

exchange

323 Remission 238

98 _{CFHS1191L Yes(2) Intoleranceto}

plasmaexchange

131 Remission 130

148a _{Unidentified No Resistanttoplasma}

exchange

415 Graftloss NS

42 _{CFH Yes(1) Resistanttoplasma}

exchange

500 Remission 62

69 _{C3R570W Yes(2) Partiallysensitiveto}

plasmaexchange 220 Remission 115 99 _CFH E3514Stop No Partiallysensitiveto plasmaexchange 565(dialysis) Remission 229

149 _{Unidentified Yes(1) Resistanttoplasma}

exchange 449(dialysis) Recurrence5 monthsfollowing withdrawalof Eculizumab.Graft loss NS 43 _{CFH No Partiallysensitiveto} plasmainfusiond 220 Remission (disappearanceof skinlesions) 209

aHUS:atypicalhaemolyticuraemicsyndrome;CFB:complementfactorBgene;CFH/CFHR1:complementhybridgeneresultingfromCFH/CFHR1

conversion;CFH:complementfactorHgene;CFHR1:complementfactorH-relatedprotein1gene;CFI:complementfactorIgene;HUS:haemolytic

uraemicsyndrome;LDH:lactatedehydrogenase;TMA:thromboticmicroangiopathy;NE:notspecified.

a _{ReceivingonlyonedoseofEculizumab.}

b _{ReduceddoseofEculizumab.}

c _{EarlyuseofEculizumab(≤7daysfollowingdiagnosisofaHUS).}

d _{SuspectedpersistenceofTMAactivityduetothepresenceofulcerativeskinlesionsinlowerlimbs.}

instudy 1treatmentwithEculizumabwas associatedwith asignificantincreaseinthenumber ofplateletsfrom base-line(p<0.001)andarateofhaematologicalnormalisationof 76%.Instudy2,80%ofpatientswerefreeofTMAepisodes following26weeksoftreatment withEculizumaband 90% hadhaematologicalnormalisation.Intermsofsecondary out-comes,treatmentwithEculizumabat26weekswasassociated withasignificantreductionintherateofdailyinterventions

for TMA vs. baseline (p<0.001), as well with a continuous improvementofestimatedGFR(+32ml/min/1.73m2_[p=0.001 vs. baseline] and +6ml/min/1.73m2 _{[p<0.001 vs. baseline]} in studies 1 and 2, respectively), adecrease inproteinuria (p<0.05) and a reduced need fordialysis. Also, the earlier Eculizumab is introduced in trials (less time of evolution between the clinical manifestationofaHUS and the drug), themoresignificanttheimprovementoftheestimatedGFR

n e f r o l o g i a. 2 0 1 5; 3 5(5) :421–447

435

Table7–MainresultsfromprospectivestudieswithEculizumabinpatientswithaHUS.

C08-002(n=17) C08-003(n=20) C10-003(n=22) C10-004(N=41)

Week26 Week64 Week100 Week26 Week62 Week156 Week26 Week26

Changeinplateletcountfrombaseline(×109_/l),mean

pvaluevs.baseline +73a <0.001 +91 0.001 +97 <0.0001 +5 NS NA NA +164 <0.0001 +135 <0.0001

Plateletcountnormalisation,b_{numberofpatients(%) 14(82) 15(88) NA NA NA NA 21(95) 40(98)}

NoTMAevents,c_{numberofpatients(%) 15(88) 15(88) 15(88) 16(80)}a _{17(85) 19(95) 21(95) 37(90)}

Haematologicalnormalisation(complete

haematologicalresponse)d_{,numberofpatients(%)}

13(76) 15(88) 15(88) 18(90) 18(90) 18(90) 18(82) 36(88)

DailyinterventionrateforTMAe_(numberof

events/patients/day)

BeforeEculizumab,mean

OnEculizumab,mean

pvaluevs.“beforeEculizumab”

0.88 0 <0.001 0.88 0 <0.001 NA 0.23 0 <0.001 0.23 0 <0.001 NA NA NA

CompleteresponseforTMA,f_{numberofpatients(%) 11(65) 13(76) NA 5(25) 7(35) NA 14(64)}a ₃₀₍₇₃₎a

OutcomeofestimatedGFR(ml/min/1.73m2₎

pvaluevs.baseline +32 0.001 +32 <0.001 +38 ≤0.05 +6 <0.001 +9 0.003 +4 NS +64 <0.0001 +29 <0.0001

Reduction≥25%inserumcreatinine,numberof

patients(%)

11(65) 13(76) 13(76) 3(15) 7(35) 11(55) 16(73) NA

IncreasedestimatedGFR≥15ml/min/1.73m2_,number

ofpatients(%)

8(47) 9(53) 10(59) 1(5) 3(15) 8(40) 19(86) 22(54)

Nodialysis,numberofpatients/numberofpatientsin

dialysisatthestartoftreatment(%)

4/5(80) 4/5(80) NA 0/2(0) 0/2(0) NA 9/11(82) 20/24(83)

ImprovementinCRDinatleastonestage,numberof

patients(%)

10(59) 11(65) 13(76) 7(35) 9(45) 12(60) 17(77) 26(63)

Reductionofproteinuriainatleastonegradein

patientswithbaselineproteinuriagrade≥1,number

ofpatients/totalnumberofpatients

12/15 9/11 NA 6/11 7/9 NA NA NA

Improvementinqualityoflife(changeinquestionnaire

scoring),meang pvaluevs.baseline +0.32 <0.001 +0.30 <0.001 +0.29 <0.0001 +0.10 <0.001 +0.13 <0.001 +0.16 ≤0.001 +19.7 <0.0001 +0.23 0.003

aHUS:haemolyticuraemicsyndrome;CRD:chronicrenaldisease;EQ-5D:EuroQoLGroup5-DimensionSelf-ReportQuestionnaire;FACIT-F:Functionalassessmentofchronicillnesstherapy-fatigue;

GFR:glomerularfiltrationrate;LDH:lactatedehydrogenase;NA:notavailable;NS:notsignificant;PE:plasmaexchange;PI:plasmainfusion;TMA:thromboticmicroangiopathy.

a _{Primaryoutcome.}

b _{Platelets≥150×10}9_/l.

c _{Noplateletdecrease>25%;noplasmatherapyandnostartofdialysisfor≥12weeks.}

d _{≥2normalconsecutivemeasurementsofplateletsandLDH,withatleast4weeksapart.}

e _{PlasmaPIorPEsessions,dialysis,orboth,perpatient/day.}

f _{Completehaematologicalresponseanddecrease≥25%inserumcreatininefrombaseline(twoconsecutivemeasurementswithatleast4weeksapart).}

g _{TheEQ-5Dquestionnairewasusedinallstudies,exceptforC10-003study,wheretheFACIT-Fquestionnaireforpaediatricpatientswasused.}