Atypical hemolytic uremic syndrome. Diana Karpman Department of Pediatrics Lund University

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1 Atypical hemolytic uremic syndrome Diana Karpman Department of Pediatrics Lund University Image courtesy of Dr. Sabine Leh, Haukeland University Hospital, Bergen Norway

2 Hemolytic Uremic Syndrome Non-immune hemolytic anemia Thrombocytopenia Acute renal failure Thrombotic microangiopathy wiki.nus.edu.sg HUS Normal

3 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

4 Classification of thrombotic microangiopathies Besbas N et al Kidney International 2006, 70: Level 1 Etiology advanced 1.i Infection induced a) Shiga and shiga-like toxin producing bacteria b) Pneumococcus 1.ii Disorders of complement regulation a) Genetic factor H, I, MCP, factor B or C3 b) Acquired i.e. anti factor H antibodies 1.iii ADAMTS13 deficiency a) Genetic b) Acquired 1.iv Defective cobalamine metabolism Level 2 Etiology unknown 2.1 HIV infection 2.ii Malignancy, cancer chemotherapy, ionizing radiation, bone marrow transplantation 2.iii Calcineurin inhibitors and transplantation 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

5 STEC-HUS Platelet Neutrophil Erythrocyte Stx Monocyte TTP C3b Atypical HUS Subendothelium C5bC6 C7C8 C9 MAC ADAMTS13 Acquired Congenital ULVWF Blood flow Schistocytes Thrombotic microangiopathies

6 Diagnostic work-up or Ariceta G et al Pediatr Nephrol 2009;24: Shiga toxin-producing bacteria Fecal culture PCR for stx, eae, uida Serum antibodies to EHEC LPS or EspB DAT negative Streptococcus pneumoniae HIV Culture from blood or CSF T antigen on RBCs agglutination of specific lectins Transferrin isoelectric focusing DAT positive Serology

7 Diagnostic work-up Complement factors and regulators Von Willebrand faktor cleaving protease ADAMTS13 Cobalamine metabolism 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 VWF cleaving activity < 5% Mutation analysis for ADAMTS13 Anti-ADAMTS13 antibodies Homocysteine, methyl malonic acid in plasma and urine. Mutations in the MMACHC gene Pregnancy, HELPP Autoimmunity Pregnancy test, LFTs Autoantibodies SLE, anti-phosholipid

8 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

9 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

10 Follow-up and treatment Platelet transfusions should be avoided Given if platelet count < 10 x10 9 /L during active bleeding or before surgical procedure

11 Atypical HUS High morbidity and mortality One study included 34 children treated in England between : 15% died and 60% developed severe complications including ESRF 21% did not develop complications and most of these had only one episode without recurrence Taylor CM et al Pediatr Nephol 2004

12 Atypical HUS pathology

13 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

14 Classical pathway Immune complex Nonimmune activators C1qr 2 s 2 Anaphylatoxin C4a C4 C2 The complement system C4b C4b2a Convertase Opsonization Lectin pathway Mannose binding lectins or ficolin binding to microbial carbohydrates MBL-MASP complex Polymeric IgA C3 Anaphylatoxin chemotaxis antimicrobial C3a C3 Alternative pathway Activating surfaces C3b C3bBb Convertase C3(H2O)Bb Immune complex binding opsonization Factor B Factor D Membrane attack complex C3a C3b Amplification loop C5 C5 Convertase Opsonization C5a C5b Anaphylatoxin chemotaxis antimicrobial C6,C7,C8,C9 MAC Kahn & Karpman APMIS 2009 Cell lysis

15 The alternative pathway C3 C3(H2O)Bb C3a C3b Factor B C3 C3bBb Convertase Factor D C3a C3b C5 Convertase C5 C5a C5b C6,C7,C8,C9 MAC Cell lysis

16 Regulation Classical pathway Lectin pathway Alternative pathway C4bp, Factor I C1INH DAF CR1 MCP C1INH DAF CR1 MCP Factor H, Factor I ic3b C4b2a Convertase DAF CR1 MCP C3bBb Convertase DAF = CD55 MCP = CD46 Factor H, Factor I C5 convertase C5 Properdin + CR1 = CD35 Protectin = CD59 C5a C5b Clusterin, S protein CD59 MAC complex

17 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

18 Mutations in atypical HUS: factor H, factor I, MCP/CD46, C3 and factor B Classical pathway Lectin pathway Alternative pathway C4bp, Factor I C1INH DAF CR1 MCP C1INH DAF CR1 MCP Factor B Factor H, Factor I Gain of function ic3b C4b2a Convertase DAF CR1 MCP C3bBb Convertase Gain of function Factor H, Factor I C5 convertase Properdin + C5 C5a C5b Clusterin, S protein CD59 MAC

19 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)

20 Regulation of the C3 convertase by soluble and cell-bound regulators Dissociation Inactivation of C3b Factor H, C4bp, DAF, CR1 Factor H, MCP, CR1 Lesher & Song Nephrology 2010

21 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

22 Factor H C3b binding C3b/C3c binding Sialic acid binding C3b/C3d binding CRP Heparin binding Heparin binding Heparin binding Heparin binding NH 2 1?? COOH 150 kda glycoprotein 20 repetitive short consensus repeats SCRs 1-20 High concentrations in human plasma: 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

23 ahus-associated mutations and polymorphisms in factor H S Rodriguez de Córdoba Clin Exp Immunol 2007

24 A model of complement activation on host endothelial cells in the presence of mutated factor H Displaced Factor B Factor H Factor D ic3b Factor I C3b C3b Glycosaminoglycans C3 convertase Factor B Factor B C3b C3b C3b C3b Normal binding of factor H to endothelial cells Reduced binding of mutated factor H to endothelial cells Vaziri-Sani F PhD thesis 2006

25 Mostly heterozygous mutations Factor H and atypical HUS 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 (C terminal) develops HUS which is C5-dependent (de Jorge EG JASN 2011)

26 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

27 Mutation database: 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

28 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

29 S Rodriguez de Córdoba Immunobiology 2012

30 CFHR1 binds to C5 and regulates the C5 convertase inhibiting MAC formation (Heinen S et al Blood 2009) Lesher & Song Blood 2009

31 Factor H related proteins 1, 2 and 5 regulate factor H

32 Endothelial cell injury Michelson AD Platelets 2002

33 Counts Factor H binding to HUVEC Control HUS Patient Vaziri-Sani F Kidney Intl 2006 Rabbit anti-goat IgG:FITC

34 Binding (%) Patients with atypical HUS and factor H mutations have excess C3 and C9 on their platelets 60 Platelets from patients and controls C3 C9 CD40L Ståhl A et al Blood 2008

35 Binding (%) Mutated factor H enables complement activation on platelets and their activation C3 C9 CD40L 30 Normal washed platelets 30 Normal washed platelets 30 Normal washed platelets Ståhl A et al Blood 2008

36 Binding (%) C3 binding to washed platelets in the presence or absence of purified factor H Ståhl A et al Blood 2008

37 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 nm in diameter Include: exosomes ( nm) shed microparticles ( nm) apoptotic bodies (1 5 µm) Mrvar-Brecko A, et al Blood Cells Mol Dis 2010

38 Microvesicles from leukocytes and platelets bear tissue factor Cambien B et al 2004 Mackman N 2004

39 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 10 3 /ml x 10 3 /ml ahus patients 631 ( ) 281 (71 521) Healthy controls 64 (41 96) 61 (42 94)

40 Summary factor H and ahus 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

41 Complement and atypical HUS Ca % of cases are associated with complement mutations/dysfunction Protein Gene Source Soluble or cellbound % 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 <5 % C3 C3 extrahepatic Soluble ~ 5-10 % Anti-FH-Abs CFHR1/ CFHR3 Lymphocytes Soluble ~ 10 % Thrombomodulin, DKGE and unknown ~ 30% Jozsi et al. 2008, Frémeaux-Bacchi V et al. 2008, Goicoechea de Jorge 2007, Caprioli, et al 2006, Kavanagh 2007

42 Treatment Plasma or plasma exchange Rituxumab For patients with auto-antibodies Soliris eculizumab (Alexion)

43 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

44 Eculizumab Soliris binds C5 inhibits terminal complement activation Patients should be vaccinated against meningococci and possible receive prophylactic antibiotics

45 Rother et al. Nat Biotech 2007;25:1256 Eculizumab: Humanized Anti - C5 Antibody Human Framework Regions No mutations Germline Hinge Complementarity Determining Regions (murine origin) CH2 Human IgG 2 Heavy Chain Constant Region 1 and Hinge (Eliminates Fc receptor binding) CH3 Human IgG 4 Heavy Chain Constant Regions 2 and 3 (Eliminates complement activation)

46 Ricklin D, et al J Immunol 2013

47 Complement and atypical HUS Risk of recurrence after renal transplantation 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 % Loirat, C et al. Pediatric Transplantation 2008, Saland et al. JASN 2009, Noris M et al, NEJM 2009

48 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 (?)

49 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

50 Effect of eculizumab after transplantation

51 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

52 Nature Reviews Nephrology 2012

53 Choice of donor

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