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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