Diana Karpman Department of Pediatrics

Lund University

Atypical hemolytic uremic syndrome

Image courtesy of Dr. Sabine Leh, Haukeland University Hospital, Bergen Norway

Hemolytic Uremic Syndrome Non-immune hemolytic anemia

Thrombocytopenia

Acute renal failure

Thrombotic microangiopathy

www.medlib.med.utah.edu

Normal HUS

wiki.nus.edu.sg

Classification

D+ HUS typical, diarrhea-associated EHEC enterohemorrhagic E. coli

STEC Shiga toxin-producing E. coli

Occurs mainly in children

Most patients (80-85%) recover without complications

Atypical HUS (aHUS) Hereditary or acquired, recurrent

Complement dysfunction: factor H, factor I, MCP, clusterin,

C3 and/or factor B mutations; thrombomodulin mutations,

anti-factor H antibodies, diacylglycerol kinase ɛ (DKGE)

mutations, drugs, cancer, autoimmune, solid organ

transplantation, pregnancy, cobalamin deficiency, idiopathic

Occurs at any age

Many progress to end stage renal failure

Classification of thrombotic

microangiopathies

Besbas N et al Kidney International 2006, 70: 423-31 Level 1 Etiology advanced Level 2 Etiology unknown

1.i Infection induced

a) Shiga and shiga-like toxin producing bacteria

b) Pneumococcus

2.1 HIV infection

1.ii Disorders of complement regulation

a) Genetic factor H, I, MCP, factor B or C3

b) Acquired i.e. anti factor H antibodies

2.ii Malignancy, cancer chemotherapy,

ionizing radiation, bone marrow transplantation

1.iii ADAMTS13 deficiency

a) Genetic

b) Acquired

2.iii Calcineurin inhibitors and transplantation

1.iv Defective cobalamine metabolism 2.iv Pregnancy HELLP syndrome,

contraceptive pill

1.v Quinine-induced 2.v Systemic lupus erythematosus,

anti-phospholipid antibody syndrome

2.vi Glomerulopathy

2.vii Familial not included in Level 1

2.viii Unclassified

Blood flow

Subendothelium

Erythrocyte

Platelet

Monocyte

Neutrophil

Schistocytes

ST

EC

-HU

S

C3b

Aty

pic

al H

US

MAC

TT

P

Congenital Acquired

C9 C8

C6

C7

C5b

ADAMTS13

ULVWF

Stx

Thrombotic

microangiopathies

Diagnostic work-up http://espn.cardiff.ac.uk/hus_guideline_2005.pdf

or

Ariceta G et al Pediatr Nephrol 2009;24:687-96

Fecal culture

PCR for stx, eae, uidA

Serum antibodies to EHEC LPS or EspB

DAT negative

Shiga toxin-producing bacteria

Serology

HIV

Culture from blood or CSF

T antigen on RBCs agglutination of specific

lectins

Transferrin isoelectric focusing

DAT positive

Streptococcus pneumoniae

Diagnostic work-up

Autoantibodies SLE, anti-phosholipid Autoimmunity

Pregnancy test, LFTs

Pregnancy, HELPP

Homocysteine, methyl malonic acid in plasma

and urine. Mutations in the MMACHC gene

Cobalamine metabolism

VWF cleaving activity < 5%

Mutation analysis for ADAMTS13

Anti-ADAMTS13 antibodies

Von Willebrand faktor cleaving

protease ADAMTS13

C3, factor H och factor I levels

Mutation analysis for factor H, FHR1 and FHR3,

factor I, MCP/CD46, factor B and C3

Anti-factor H antibodies

Complement factors and regulators

Laboratory findings

• Anemia

• Hemolysis: elevated LD, bilirubin, reticulocytes,

decreased haptoglobin, fragmented RBCs

• DAT negative

• Thrombocytopenia. Normal PK, APTT

• Renal failure: elevated urea, creatinine,

potassium and acidosis

Follow-up and treatment

Weight

Fluid intake and urinary output

Hydration IV w/o potassium

Anti-hypertensive treatment: Loop diuretic, Nifedipine,

Labetolol, Clonidine

Hyperkalemia and acidosis

Anti-epileptics

Dialysis: hypervolemia, hyperkalemia, acidosis, uremia

Nutrition: carbohydrates with essential amino acids

Hb < 60 blood transfusion

Platelet transfusions should be avoided

Given if platelet count < 10 x109/L

during active bleeding or before surgical procedure

Follow-up and treatment

Atypical HUS

Taylor CM et al Pediatr Nephol 2004

High morbidity and mortality

One study included 34 children treated in England between1998-99:

15% died and 60% developed severe complications

including ESRF

21% did not develop complications and most of these had

only one episode without recurrence

Atypical HUS pathology

Complement

• fights infection

• removes damaged host cells

• modulates adaptive immunity

• Identification of a foreign antigen/microorganism/unwanted cell

• Labeling (opsonisation) of the foreign /unwanted particle

• Killing or damaging the foreign bacteria or apoptotic cell

Classical pathway Lectin pathway

Immune complex

Nonimmune activators

Activating surfaces

Alternative pathway

Anaphylatoxin

Anaphylatoxin

chemotaxis

antimicrobial

Opsonization

Opsonization

Amplification loop

Anaphylatoxin

chemotaxis

antimicrobial

Mannose binding lectins or ficolin binding to

microbial carbohydrates

Polymeric IgA

C1qr2s2 C4

MBL-MASP complex

C4a C4b

C2

C3a C3b

C3

C3

C3a C3b

Factor B

Factor D

C5

C5a

C5b

C6,C7,C8,C9

MAC

C3(H2O)Bb

C5 Convertase

Cell lysis

C4b2a Convertase C3bBb Convertase

Membrane attack complex

Kahn & Karpman APMIS 2009

Immune complex binding

opsonization

The complement system

The alternative pathway

C3a C3b

C3

C3

C3b

Factor B

Factor D

C5

C5a

C5b

C6,C7,C8,C9

MAC

C5 Convertase

Cell lysis

C3bBb Convertase

C3a

C3(H2O)Bb

Regulation

Classical pathway

C4b2a Convertase

Alternative pathway

C3bBb Convertase

MAC complex

C5 convertase

Lectin pathway

C4bp, Factor I

C1INH C1INH Factor H, Factor I

Clusterin, S protein

Factor H, Factor I

Properdin +

iC3b

DAF

CR1

MCP

DAF

CR1

MCP

CD59

C5

C5a C5b

DAF

CR1

MCP

DAF = CD55

MCP = CD46

CR1 = CD35

Protectin = CD59

Mechanisms of complement activation via

the alternative pathway in

atypical HUS

• Mutated complement regulators with loss-of-function

• Gain-of-function mutations in complement factors

• Autoantibodies to complement regulator

Mutations in atypical HUS:

factor H, factor I, MCP/CD46, C3 and factor B Classical pathway

C4b2a Convertase

Alternative pathway

C3bBb Convertase

MAC

C5 convertase

Lectin pathway

C4bp, Factor I

C1INH C1INH Factor H, Factor I

Clusterin, S protein

Factor H, Factor I

Properdin +

iC3b

DAF

CR1

MCP

DAF

CR1

MCP

CD59

C5

C5a C5b

DAF

CR1

MCP

Factor B Gain of function

Gain of function

Factor H disease associations

Dysregulation of the alternative pathway due to mutations or polymorphisms:

• Atypical hemolytic uremic syndrome

• Membranoproliferative glomerulonephritis (MPGN) type II (Dense deposit disease)

• Age-related macular degeneration (AMD)

Regulation of the C3 convertase by soluble

and cell-bound regulators

Lesher & Song Nephrology 2010

Dissociation Inactivation of C3b

Factor H, MCP, CR1 Factor H, C4bp, DAF, CR1

Complement activation via the alternative pathway

on foreign surfaces

The C terminal of factor H and host cell recognition

Vaziri-Sani F PhD thesis 2006

Factor H

7 9 13 20 1 NH2 COOH

C3b binding

Heparin binding

C3b/C3c binding C3b/C3d binding

Heparin binding Heparin binding Heparin binding

? ?

150 kDa glycoprotein

20 repetitive short consensus repeats SCRs 1-20

High concentrations in human plasma: 110 - 560 µg/ml

Inhibits activation of C3, regulates the alternative pathway

Cofactor for complement factor I in cleaving C3b to iC3b (N terminal)

Prevents formation of the C3bBb convertase

Accelerates decay of C3Bb convertase (N terminal)

Discriminates between host and foreign cells (C terminal)

by the presence of polyanion molecules on host cells

Sialic acid binding

CRP

aHUS-associated mutations and polymorphisms

in factor H

S Rodriguez de Córdoba Clin Exp Immunol 2007

A model of complement activation on host endothelial

cells in the presence of mutated factor H

Vaziri-Sani F PhD thesis 2006

Normal binding of factor H to endothelial cells

C3b iC3b

Factor I

C3b

Displaced Factor B

Factor H

Reduced binding of mutated factor H to endothelial cells

Glycosaminoglycans

C3 convertase

Factor D

C3b

Factor B

C3b C3b C3b

Factor B

Factor H and atypical HUS

• Mostly heterozygous mutations

• Disease-associated polymorphisms

• May co-exist with mutations in other complement regulators

• Most aHUS patients have normal levels of C3 and factor H

• Normal factor H activity in plasma but not on cells

• Normal co-factor activity for factor I-mediated cleavage of C3

• Incomplete penetrance. Genetic and environmental factors contribute

• A mouse model with a deletion in SCRs 16-20 (C terminal)

develops HUS which is C5-dependent (de Jorge EG JASN 2011)

Antibodies to factor H

Directed to the C terminal

may be associated with rearrangements in factor H-related proteins

Zipfel P et al Pediatr Nephrol 2010

Mutation database: http://www.fh-hus.org

S Rodriguez de Córdoba Clin Exp Immunol 2007

Patients with anti-factor H antibodies may have a homozygous

Deletion or rearrangements of the CFHR genes

Factor H gene

Located on chromosome 1q32

in the regulator of complement activation (RCA) gene cluster

Factor-H like 1 FHL-1 protein 43 kD consists of SCRs 1-7

Five factor H-related FHR proteins consisting of 4-9 SCRs

SR de Córdoba Clin Exp Immunol 2007

S Rodriguez de Córdoba Immunobiology 2012

Lesher & Song Blood 2009

CFHR1 binds to C5 and regulates the C5 convertase

inhibiting MAC formation

(Heinen S et al Blood 2009)

http://bloodjournal.hematologylibrary.org/content/114/12/2363/F1.expansion.html

Factor H related proteins 1, 2 and 5

regulate factor H

Endothelial cell injury

Michelson AD Platelets 2002

80

40

60

0

20

100

Counts

Rabbit anti-goat IgG:FITC

Factor H binding to HUVEC

Control HUS Patient

Vaziri-Sani F Kidney Intl 2006

Patients with atypical HUS and factor H mutations

have excess C3 and C9 on their platelets

Platelets from patients

and controls

0

10

20

30

40

50

60

C3 C9 CD40L

Bin

din

g (

%)

Ståhl A et al Blood 2008

Bin

din

g (

%)

C3

Normal washed platelets

C9

Normal washed platelets

CD40L

Normal washed platelets

0

5

10

15

20

25

30

0

5

10

15

20

25

30

0

5

10

15

20

25

30

Mutated factor H enables complement activation

on platelets and their activation

Ståhl A et al Blood 2008

C3 binding to washed platelets in the presence

or absence of purified factor H

0

10

20

30

40

50

60

Bin

din

g (

%)

Ståhl A et al Blood 2008

Microvesicles

• Extracellular organelles shed from

cells during activation or apoptosis

• Contain proteins, RNA, miRNA, DNA and histones

• Express markers or contents of the parent cell

• 40 – 5000 nm in diameter

• Include:

– exosomes (40 – 100 nm)

– shed microparticles (100 – 1000 nm)

– apoptotic bodies (1 – 5 µm)

Mrvar-Brecko A, et al

Blood Cells Mol Dis 2010

Cambien B et al 2004

Microvesicles from leukocytes and platelets

bear tissue factor

Mackman N 2004

http://atvb.ahajournals.org/content/vol24/issue6/images/large/6FF3.jpeg

Tissue factor expression after exposure of normal

washed platelets to aHUS patient sera

Ståhl A Blood 2008

Serum Tissue factor Tissue factor positive

positive platelet platelet microvesicles

microvesicles after exposure to factor H

x 103/mL x 103/mL

aHUS patients 631 (128 - 897) 281 (71 – 521)

Healthy controls 64 (41 – 96) 61 (42 – 94)

• Mutated factor H allows complement activation

to occur on endothelial cells and platelets

• Mutated factor H promotes tissue factor

expression on platelet microvesicles

• Complement activation results in endothelial cell injury,

platelet activation and a prothrombotic state

Summary factor H and aHUS

Complement and atypical HUS

Ca 60-70 % of cases are associated with complement

mutations/dysfunction

Protein Gene Source Soluble or cell-

bound

% of aHUS

Factor H CFH Liver Soluble ~ 30 %

Factor I CFI Liver Soluble ~ 10 %

Membrane cofactor

protein/CD46

MCP Many cells Cell-bound ~ 15 %

Factor B CFB Liver and Soluble

Treatment

Plasma or plasma exchange

Rituxumab

For patients with auto-antibodies

Soliris eculizumab (Alexion)

Plasma exchange or infusion? Sakari Jokiranta et al Mol Immunol 2007

• Plasma infusion can lead to increased colloid pressure

and hypertension in patients with renal failure

• Plasma exchange will replace mutated complement factors

• Patients with MCP and DKGE mutations should theoretically

not benefit from plasma

Eculizumab Soliris

binds C5 inhibits terminal complement activation

Patients should be vaccinated against meningococci

and possible receive prophylactic antibiotics

Eculizumab:

Humanized Anti - C5 Antibody

Hinge

CH

3

CH

2

Human IgG4 Heavy Chain

Constant Regions 2 and 3

(Eliminates complement activation)

Complementarity Determining Regions

(murine origin)

Human Framework Regions

• No mutations

• Germline

Human IgG2 Heavy Chain

Constant Region 1 and Hinge

(Eliminates Fc receptor binding)

Rother et al. Nat Biotech 2007;25:1256

Ricklin D, et al

J Immunol 2013

Risk of recurrence after renal transplantation

Loirat, C et al. Pediatric Transplantation 2008, Saland et al. JASN 2009, Noris M et al, NEJM 2009

Protein Gene Source Soluble/

Cell bound

Risk of

recurrence

Factor H CFH Liver Soluble ~ 80 %

Factor I CFI Liver Soluble ~ 80 %

Membrane cofactor

protein/CD46

MCP Many cells Cell-bound ~ 20 %

Factor B CFB Liver/extrahepatic Soluble Recurs

C3 C3 Liver/extrahepatic Soluble ~ 50%

Anti-FH-Abs CFHR1/

CFHR3

Lymphocytes Soluble ~ 20%

Unknown ~ 30 %

Complement and atypical HUS

Renal transplant

Ca 50 % of aHUS cases recur after transplantation

Close to 100 % of cases with factor H or factor I mutations

Better prognosis if only MCP mutation

Avoid living-related donor (?)

Eculizumab for aHUS transplantation

22 transplanted aHUS patients:

9 treated preemptively with Eculizumab, 8 with good tx function

13 treated after recurrence also with good effect

Effect of eculizumab after transplantation

Zuber J et al Am J Transpl 2012

• Eculizumab was effective for aHUS de

novo as well as for recurrence after

transplantation

• Treatment should be commenced ASAP

after recurrence

• Prolonging treatment intervals increases the

risk of recurrences

Nature Reviews Nephrology 2012

Choice of donor

diana.karpman@med.lu.se