Thrombotic Microangiopathies

T. Matthew Eison, MD
LeBonheur Children’s Hospital
Pediatric Nephrology
 TMA Timeline
-1924: Moschcowitz presented first published example of
idiopathic TTP before New York Pathological Society
[1,2]
-1952: Thrombotic Microangiopathy first described in by
Symmers [3]
-1965: Familial Atypical HUS first described in
combination of hemolytic anemia & azotemia in
concordant monozygous twins [4]
-1974: Reduced serum levels of complement fraction C3
with normal levels of C4 reported in patients with atypical
HUS[5-7]
-1976: Bukowski et al published experience w/whole
blood exchange transfusion, w/8 /14 TTP patients
w/TTP achieving prompt remissions lasting several
months to >13 yrs[8]
-1981: investigators described two brothers with atypical
HUS w/complete complement factor H (CFH) deficiency
[9]; parents, who were first cousins, had half-normal
CFH levels, indicating inherited defect.
-1982: Moake linked von Willebrand factor (VWF) to the
pathogenesis of TTP, recognizing circulating “ultralarge”
VWF multimers in relapsing acquired or congenital TTP
patients in remission [10]
1. Moschcowitz E. 1924;24:21-24.
2. Moschcowitz E. Arch Intern Med. 1925;36:89-93
3. Symmers WStC: Br Med J 2:897–903, 1952
4. Campbell S, Carré IJ. Arch Dis Child 1965;40:654-8.
5. Carreras L, Romero R, Requesens C, et al. JAMA 1981;245:602-4.
6. Stühlinger W, Kourilsky O, Kanfer A, Sraer JD. Lancet 1974;2:788-9.
7. Noris M, Ruggenenti P, Perna A, et al. J Am Soc Nephrol 1999;10:281-93.
8. Bukowski RM, Hewlett JS, Harris JW, et al. Semin Hematol. 1976;13:219-232.
9. Thompson RA, Winterborn MH. Clin Exp Immunol 1981;46:110-9.
10. Moake JL, Rudy CK, Troll JH, et al. N Engl J Med. 1982;307: 1432-1435.
11. Tsai H-M. Blood. 1996;87:4235-4244.
-1996: candidate VWF-cleaving protease requiring fluid
shear stress or low protein denaturant concentration
identified in human plasma by Tsai [11] & independently
by Furlan [12]
-1997: VWF-cleaving protease demonstrated missing
from congenital TTP patients' plasma [13]
-1998: Goodship ‘s investigative group [14] showed
association b/w atypical HUS & chromosome 1q32
locus, containing genes for CFH and other complement
regulators
-1998: Adults with acquired idiopathic TTP reported to
have severe VWF-cleaving protease deficiency caused
by IgG autoantibodies that inhibit the enzyme [15,16]
-2001: VWF-cleaving protease purified, cloned, and
named ADAMTS13 [17-22] because it belonged to the
recently discovered “a disintegrin-like and
metalloprotease metalloprotease with thrombospondin
repeats” family of metalloproteases [24]
-2001: Mutations in the ADAMTS13 gene shown to
cause congenital TTP [21]
12. Furlan M, Robles R, La¨mmle B. Blood.1996;87:4223-4234.
13. Furlan M, Robles R et al. Blood. 1997;89:3097-3103.
14. Warwicker P, Goodship TH, Donne RL, et al. Kidney Int 1998;53:836-44.
15. Tsai HM, Lian EC. N Engl J Med. 1998; 339:1585-1594.
16. Furlan M, Robles R, Galbusera M, et al. N Engl J Med. 1998;339:1578- 1584.
17. Fujikawa K, Suzuki H, McMullen B, Chung D. Blood. 2001;98: 1662-1666.
18. Gerritsen HE, Robles R, Lammle B, Furlan M. Blood. 2001;98:1654-1661.
19. Soejima K, Mimura N, Hirashima M, et al. J Biochem. 2001;130:475-480.
20. Zheng X et al. J Biol Chem. 2001;276: 41059-41063.
21. Levy GG, Nichols WC, Lian EC, et al. Nature. 2001;413:488-494.
22. Hurskainen TL, Hirohata S, Seldin MF, Apte SS. J Biol Chem. 1999;274:25555-25563.
Noris M, Remuzzi G. Hemolytic uremic syndrome. J Am Soc Nephrol. 2005 Apr;16(4):1035-50.
 Thrombotic Microangiopathy
-pathological term used to describe occlusive microvascular thrombus formation 1
-first described in 1952 by Symmers 2
-Pathological features:
--vessel wall thickening
--swelling and detachment of the endothelial cell from the basement membrane
--accumulation of material in the subendothelial space
--intraluminal platelet thrombosis
--partial or complete vessel luminal obstruction and fragmentation of red blood cells 3–6

1. Zheng XL, Sadler JE (2008) Pathogenesis of thrombotic microangiopathies. Annu Rev Pathol 3:249–277
2. Symmers WStC: Thrombotic microangiopathic haemolytic anemia (thrombotic microangiopathy). Br Med J 2:897–903, 1952
3. Ruggenenti P, Noris M, Remuzzi G (2001) Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic
purpura. Kidney Int 60:831–846
4. Copelovitch L, Kaplan BS (2008) The thrombotic microangiopathies. Pediatr Nephrol 23:1761–1767
5. Tsai HM (2006) The molecular biology of thrombotic microangiopathy. Kidney Int 70:16–23
6. Benz K, Amann K (2009) Pathological aspects of membranoproliferative glomerulonephritis (MPGN) and haemolytic uraemic syndrome
(HUS)/thrombocytic thrombopenic purpura (TTP).Thromb Haemost 101:265–270
Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular
thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
RBCs and fibrin fill up capillary loops in glomerulus in thrombotic microangiopathy
(Jones’ silver stain) –Agnes Fogo
Fibrin thrombi extending from glomerulus into arteriole in thrombotic microangiopathy
(Jones’ silver stain) –Agnes Fogo
Fibrin tactoids in subendothelial area by EM in thrombotic microangiopathy. –Agnes Fogo
 Thrombotic Microangiopathy:
Clinical Asociations
--consumptive thrombocytopenia
--microangiopathic hemolytic anemia
--features of organ ischemia1
--symptoms produced depend on the vascular bed and organ affected
--most commonly associated with Hemolytic Uremic Syndrome (HUS)
and Thrombotic Thrombocytopaenic Purpura (TTP)2

1. Tsai HM (2006) The molecular biology of thrombotic microangiopathy. Kidney Int 70:16–23
2. Zheng XL, Sadler JE (2008) Pathogenesis of thrombotic microangiopathies.
Annu Rev Pathol 3:249–277
 Is it TTP or HUS?
--Hemolytic uremic syndrome (HUS):
TMA predominantly seen in the glomeruli; results in acute renal insufficiency *
--Thrombotic Thrombocytopenic Purpura (TTP):
neurological endothelial cells predominantly affected;
therefore neurological features seen*
--TTP and HUS often considered together:
----similar etiology
----overlap in clinical symptoms
(neurological features in HUS; renal problems in TTP)
----many patients best described as TTP-HUS (Up To Date):
severe neurologic sx (seizures & coma) with acute renal failure;

*Tsai HM (2006) The molecular biology of thrombotic microangiopathy. Kidney Int

70:16–23
 Alternate TTP vs HUS Classification

George JN. The thrombotic thrombocytopenic purpura and hemolytic uremic syndromes: overview of
pathogenesis (Experience of The Oklahoma TTP-HUS Registry, 1989-2007). Kidney Int Suppl. 2009 Feb;
(112):S8-S10.
Besbas N et al; European Paediatric Research Group for HUS. A classification of hemolytic uremic syndrome and
thrombotic thrombocytopenic purpura and related disorders. Kidney Int. 2006 Aug;70(3):423-31
Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular
thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
Ruggenenti P, Noris M, Remuzzi G (2001) Thrombotic microangiopathy, hemolytic uremic syndrome,
and thrombotic thrombocytopenic purpura. Kidney Int 60:831-46.
 Thrombotic Thrombocytopenic Purpura:
A New Disease Discovery
-In Jan.1 & Feb. 1924 2 Moschcowitz presented
1st known case before
New York Pathological Society:
“a hitherto undescribed disease”
“remarkable, clinically and anatomically”
-Case: Healthy 16-year-old girl w/initial sx
----sudden arm weaknessarms
----pain w/wrist and elbow movement
----pallor
----fever (38°C-39°C)
-Symptoms worsened; Day #10 illness, admitted
w/findings
----anemia
----leukocytosis
----a few petechiae on one arm
----occult blood in gastric contents and stool
----nl serum creatinine
1. Moschcowitz E. Proc N Y Pathol Soc. 1924;24:21-24.
2. Moschcowitz E. Arch Intern Med. 1925;36:89-93.
3. Sadler JE. Blood. 2008 Jul 1;112(1):11-8.
-Day #14 illness:
----mild left hemiparesis and facial paralysis
-Day #15 illness:
----became comatose and died
-On limited autopsy: hyaline thrombi in terminal
arterioles and capillaries of heart, kidney,
spleen, and liver; the lungs were spared
-No platelet count obtained;
-no report of schistocytes;
-therefore not complete description of
thrombocytopenia or
microangiopathic hemolytic anemia
(Nonetheless, recognized as first published
example of idiopathic TTP)
 Thrombotic Thrombocytopenic Purpura (TTP):
Epidemiology

-more common in females age 10-39 yrs

-highest incidence is seen in fourth decade
-annual incidence 3.7 cases per 1,000,0001

1. Michael M, Elliott EJ, Ridley GF, Hodson EM, Craig JC (2009) Interventions for haemolytic uraemic
syndrome and thrombotic thrombocytopenic purpura. Cochrane Database Syst Rev CD003595
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Thrombotic Thrombocytopenic Purpura (TTP):
Clinical Presentation
-Classic "Pentad" of symptoms:
----microangiopathic hemolytic anemia
----thrombocytopaenia
----neurological symptoms
----renal damage
----fever
-Previously a diagnosis of exclusion; now ADAMTS13 activity assay in use1
-Plasma exchange improves symptoms; now standard treatment for TTP2

1. Sadler JE (2008) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura.
Blood 112:11–18
2. Zheng XL, Sadler JE (2008) Pathogenesis of thrombotic microangiopathies. Annu Rev Pathol 3:249–277
3. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
George JN. The thrombotic thrombocytopenic purpura and hemolytic uremic syndromes: overview of
pathogenesis (Experience of The Oklahoma TTP-HUS Registry, 1989-2007). Kidney Int Suppl. 2009 Feb;
(112):S8-S10.
Vesely SK, George JN, Lämmle B, Studt JD, Alberio L, El-Harake MA, Raskob GE. ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic
uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood. 2003 Jul 1;102(1):60-8
George JN. The thrombotic thrombocytopenic purpura and hemolytic uremic syndromes: overview of
pathogenesis (Experience of The Oklahoma TTP-HUS Registry, 1989-2007). Kidney Int Suppl. 2009 Feb;
(112):S8-S10.
 Complications of Plasma Exchange in TTP

In a study of 57 consecutive patients from 2002-2005 from the

Oklahoma TTP-HUS Registry:

Howard MA, Williams LA, Terrell DR, Duvall D, Vesely SK, George JN. Complications of plasma exchange in patients treated
for clinically suspected thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. Transfusion. 2006 Jan;46(1):154-
6.
 Complications of Plasma Exchange in TTP

Howard MA, Williams LA, Terrell DR, Duvall D, Vesely SK, George JN. Complications of plasma exchange in patients treated
for clinically suspected thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. Transfusion. 2006 Jan;46(1):154-
6.
 Thrombotic Thrombocytopenic Purpura (TTP):
History of Pathogenesis Elucidation
-TTP treated successfully x 20 yrs w/plasma exchange before mechanism
understood1
-In 1982, TTP patients recognized to have circulating ultra-large multimers of
von Willebrand factor during remission
(as multimers not in healthy people, TTP patients were hypothesized to lack
protease to cleave ultra-large multimers)2
-TTP now known to be disorder of von Willebrand factor (vWF) regulation3

1. Moake J (2009) Thrombotic thrombocytopenia purpura (TTP) and other thrombotic microangiopathies.
Best Pract Res Clin Haematol 22:567–576
2. Sadler JE (2008) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood
112:11–18
3. Tsai HM (2006) The molecular biology of thrombotic microangiopathy. Kidney Int 70:16–23
4. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Thrombotic Thrombocytopenic Purpura (TTP) Pathogenesis:
von Willebrand factor (vWF)

-glycoprotein produced by endothelial cells

-regulates platelet aggregation and adhesion
-released from endothelial cells as ultralarge multimers (UL-vWF) when vascular injury
occurs
-Some multimers remain associated with endothelial cell surface
(providing platelet-binding sites)
-Multimers may bind other blood components too, (e.g. leukocytes)
-Platelet binding to UL-vWF regulated by the metalloprotease ADAMTS13
(a-disintegrin-like and metalloprotease and thrombospondin repeats)
-deficiency of ADAMTS 13 accounts for majority of patients with congenital TTP
-majority of acquired cases occur due to antibody formation against ADAMTS 13 1

1. Sadler JE (2008) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood
112:11–18
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
Sadler JE. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura.
Blood. 2008 Jul 1;112(1):11-8.
Sadler JE. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura.
Blood. 2008 Jul 1;112(1):11-8.
Vesely SK, George JN, Lämmle B, Studt JD, Alberio L, El-Harake MA, Raskob GE. ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic
uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood. 2003 Jul 1;102(1):60-8
 Hemolytic uremic syndrome (HUS):
Epidemiology
-More commonly seen in children rather than adults
-most common cause of pediatric acute renal failure
-occurs most frequently in children under the age of 5 years
-annual incidence of 6.1 cases per 100,000 children under 5 years
-affects 0.2 - 4.28 people per 100,000 worldwide1

1. Serna A 4th, Boedeker EC (2008) Pathogenesis and treatment of Shiga toxin-producing

Escherichia coli infections. Curr Opin Gastroenterol 24:38–47
2. Noris M, Remuzzi 1. G. Hemolytic uremic syndrome. J Am Soc Nephrol 2005; 16:1035-50
 Hemolytic Uremic Syndrome Classification
-A) Typical
-More than 90% of cases in children
-secondary to infection with
---1) Escherichia coli serotypes O157:H7, O111:H8, O103:H2, O123, O26,
or others, which produce Shiga-like toxin (Stx) 1
---2) several other bacteria, such as Streptococcus pneumoniae. 2
-B) Atypical
-Approximately 10% of cases in children
-not caused by either Stx-producing bacteria or streptococci. 1,3
-poor prognosis:
---1) death rates as high as 25%3
---2) progression to end-stage renal disease in half the patients. 1,2
-Research has linked to uncontrolled activation of the complement system.
-more commonly associated with extra-renal effects than typical HUS 4

*Stx-related lesions indistinguishable from atypical form via standard histologic analysis. 4,5

1. Noris M, Remuzzi 1. J Am Soc Nephrol 2005; 16:1035-50.

2. Constantinescu AR, Bitzan M, Weiss LS, et al. Am J Kidney Dis 2004;43:976-82.
3. Kaplan BS, Meyers KE, Schulman SL. J Am Soc Nephrol 1998;9:1126-33.
4. Ruggenenti P, Noris M, Remuzzi G. Kidney Int 2001;60: 831-46.
5. Benz K, Amann K. Thromb Haemost 2009;101:265-70.
 Shigatoxin/Diarrhea-associated HUS:
Epidemiology
-Most common cause of HUS1
-predominantly a disease of childhood, but can also affect adults, particularly the elderly
-It is fatal in 3–5% of cases
-overall incidence 2.1 cases per 100,000 persons/yr 2
-peak incidence in children younger than 5 yr (6.1 per 100,000/yr) 2
-lowest incidence in adults 50 to 59 yr of age (0.5 per 100,000/yr) 2
-incidence parallels seasonal fluctuation of E. coli O157:H7 infections
[peak in warmer months (June-September)]
-In U.S., causes approx. 70,000 illnesses & 60 deaths annually 3
-In Argentina & Uruguay, E. coli infections endemic; estimated Stx-HUS incidence 10.5 per
100,000/yr4

1. Scheiring J, Andreoli SP, Zimmerhackl LB (2008) Pediatr Nephrol 23:1749–1760

2. Ruggenenti P, Noris M, Remuzzi G: Kidney Int 60: 831–846, 2001
3. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV: Emerg Infect Dis 5: 607–625, 1999
4. Meichtri L, Miliwebsky E, Gioffre A, Chinen I, Baschkier A, Chillemi G, Guth BE, Masana MO, Cataldi A, Rodriguez HR,
Rivas M: Int J Food Microbiol 96: 189–198, 2004
5. Keir L, Coward RJ. Pediatr Nephrol. 2011 Apr;26(4):523-33
6. Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50.
 Shigatoxin/Diarrhea-associated HUS:
Source of Infection
Vectors for Stx-producing E. coli transmission1,2 : Sources of infection in humans
---Cattle (colonize healthy cattle intestine) ---manure and water troughs in farms (therefore
---deer increased risk of infection in rural areas)
---sheep ---contaminated milk
---goats ---contaminated meat (contamination occurs at
---horses slaughter; bacteria internalized during grinding;
survives cooking)3
---dogs
---unchlorinated water4
---birds
---contact with infected animals or humans
---flies excrement3,5,6
---environmental contamination7
---Contaminated fruits and vegetables (radish
sprouts, lettuce, apple cider)
---Unpasteurized apple juice in several outbreaks8
---Person-to-person (child care and long-term care
facilities)3

1. Ruggenenti P, Noris M, Remuzzi G: Kidney Int 60: 831–846, 2001
2. Griffin PM, Tauxe RV: Epidemiol Rev 13: 60–98, 1991
3. Mead PS, Griffin PM: Lancet 352: 1207–1212, 1998
4. McCarthy TA et al: Pediatrics 108: E59, 2001
5. Locking ME et al: Epidemiol Infect 127: 215–220, 2001
6. Mead PS et al: Arch Intern Med 157: 204–208, 1997
7. Varma JK et al: JAMA 290: 2709–2712, 2003
8. Cody SH et al: Ann Intern Med 130: 202–209, 1999
9. Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50
 Shigatoxin/Diarrhea-associated HUS:
Clinical Presentation
-Avg interval E. coli exposure to illness 3 days (range, 1 to 8)1
-Typical initial presentation of abdominal cramps & nonbloody diarrhea
-Diarrhea hemorrhagic in 70% of cases; usually within 1 or 2 days 1
-2 to 5 days post diarrhea onset with symptoms of pallor, weakness, oligo-anuria
-HUS usually diagnosed 6 days post diarrhea onset2
-Symptom Associations: Vomiting (30-60%); fever (30%), elevated WBC
-Barium Enema: “thumb-printing” (edema & submucosal hemorrhage, especially in
ascending & transverse colon)2

1. Chandler WL, Jelacic S, Boster DR, Ciol MA, Williams GD, Watkins SL, Igarashi T, Tarr PI: Prothrombotic coagulation
abnormalities preceding the hemolytic-uremic syndrome. N Engl J Med 346: 23–32, 2002
2. Ruggenenti P, Noris M, Remuzzi G: Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic
thrombocytopenic purpura. Kidney Int 60: 831–846, 2001
3. Keir L, Coward RJ. Pediatr Nephrol. 2011 Apr;26(4):523-33
4. Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50 .
 Shigatoxin/Diarrhea-associated HUS:
Risk of Developing HUS
-Only 10–15% of enterohaemorrhagic E. coli- infected children develop HUS 1
-Associated with increased risk of HUS post E. coli infection2:
---diarrhea
---fever
---vomiting
---elevated leukocyte count
---extremes of age
---female gender
---antimotility agents3

1. Keir L, Coward RJ. Pediatr Nephrol. 2011 Apr;26(4):523-33

2. Mead PS, Griffin PM: Escherichia coli O157:H7. Lancet 352: 1207–1212, 1998
3. Beatty ME, Griffin PM, Tulu AN, Olsen SJ: Culturing practices and antibiotic use in children with diarrhea. Pediatrics 113:
628–629, 2004
4. Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50
 Shigatoxin/Diarrhea-associated HUS:
Diagnosis
-Stx-E. coli may shed in stool several weeks post symptoms resolution,
particularly in children <5 yrs of age1
-Diagnosis depends on stool culture-detection of Stx-E. coli
-Research laboratories can perform serologic tests for Stx- and O157 LPS-
antibodies
-Rapid detection tests of stool for E. coli O157:H7 & Stx being developed

1) Ruggenenti P, Noris M, Remuzzi G: Thrombotic microangiopathy, hemolytic uremic syndrome, and

thrombotic thrombocytopenic purpura. Kidney Int 60: 831–846, 2001
2) Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50.
 Shigatoxin/Diarrhea-associated HUS:
Sequelae
-50-75% diarrhea-associated HUS (D + HUS) cases require dialysis therapy in the acute phase 1
-70% of HUS patients require RBC transfusions
-25% have neurologic involvement, including stroke, seizure, and coma 2,3,4
-Despite ICU & Dialysis, 3-5% of Stx-HUS patients die in acute phase 2
-12% risk of death or permanent ESRD, 25% risk of GFR < 80 ml/min/1.73 m2
(Meta-analysis of 3476 patients from 49 published studies, mean follow-up 4.4 yr) 4
-Associated with poor long-term prognosis:
severe acute illness, CNS symptoms, need for initial dialysis 4,5
-Mortality rate ~ 30% when associated with S. dysenteriae infection 6
(often complicated by bacteremia w/septic shock, DIC, acute cortical necrosis)

1. Scheiring J, Andreoli SP, Zimmerhackl LB (2008) Pediatr Nephrol 23:1749–1760

2. Milford D: The hemolytic uremic syndromes in the United Kingdom. In: Hemolytic Uremic Syndrome and Thrombotic
Thrombocytopenic Purpura, edited by Kaplan BS, Trompeter RS, Moake JL, New York, Marcel Dekker, 1992, pp 39–59
3. Mead PS, Griffin PM: Escherichia coli O157:H7. Lancet 352: 1207–1212, 1998
4. Garg AX, et al. JAMA 290: 1360–1370, 2003
5. Tonshoff B, Sammet A, Sanden I, Mehls O, Waldherr R, Scharer K: Nephron 68: 63–70, 1994
6. Date A, Raghupathy P, Jadhav M, Pereira SM, Shastry JC: Ann Trop Paediatr 2: 1–6, 1982
7. Keir L, Coward RJ. Pediatr Nephrol. 2011 Apr;26(4):523-33
8. Noris M, Remuzzi G. J Am Soc Nephrol. 2005 Apr;16(4):1035-50.
 Shigatoxin/Diarrhea-associated HUS:
Pathogenesis
-Stx-producing serotypes (E. coli O157:H7, O111, nonmotile O26:H11, O103:H2) may produce
large amts of Stx
-Stx traverse polarized gastrointestinal epithelial cells via transcellular pathways 1,
to systemic circulation
(However, Free toxin has never been detected HUS patient's circulation)
-Shiga toxin's high-affinity cellular target: Globotriaosylceramide (Gb3)2
-Gb3: glycosphingolipid receptor on human endothelial cells, podocytes and tubular cells
-Podocytes, proximal tubule cells, glomerular endothelial cells all sensitive to Stx cytotoxicity.
-In vitro, Stx rapidly binds with low-affinity to polymorphonuclear leukocytes (PMNs) (not
erythrocytes, monocytes, platelets, or lipoproteins)3
-PMN Stx receptor 100-fold lower affinity than high-affinity receptor Gb3 expressed on
glomerular endothelial cells.
-in in vitro cocultures, PMNs Stx to glomerular endothelial cells, which promote cell death 3
(in vivo would explain rapid clearance of Stx from circulation and release to target organs)
1. AchesonDWK,MooreR,DeBreuler S: Infect Immunol 64:3294–3300, 1996
2. Psotka MA, Obata F, Kolling GL, Gross LK, Saleem MA, Satchell SC, Mathieson PW, Obrig TG (2009) Shiga toxin 2 targets the murine
renal collecting duct epithelium. Infect Immun 77:959–969
3. te Loo DMWM, Monnens LAH, van der Velden TJAM, et al: Blood 95:3396–3402, 2000
4. Ruggenenti P, Noris M, Remuzzi G. Kidney Int. 2001 Sep;60(3):831-46.
5. Keir L, Coward RJ. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Streptococcal pneumoniae-related HUS:
Epidemiology
-accounts for 5% of childhood cases of HUS1
-incidence of HUS following pneumococcal infection estimated to be
0.4–0.6%
-Children under 2 years are most commonly affected

1. Copelovitch L, Kaplan BS (2008) Streptococcus pneumoniae associated hemolytic uremic syndrome.

Pediatr Nephrol 23:1951– 1956
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Streptococcal pneumoniae-related HUS:
Clinical Presentation
-follows invasive pneumococcal disease
-linked to having a high bacterial load
-HUS usually develops 3–13 days post initial infection
-associated with a longer period of oligoanuria & acute dialysis period than stx
HUS
-10% of patients progress to ESRD; 12% mortality
-highest mortality related to S.pneumoniae meningitis complicated by HUS

Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Streptococcal pneumoniae-related HUS Pathogenesis:
Thomsen–Friedenreich (TF) crypt antigen

-found on surface of erythrocytes, platelets and glomerular endothelial cells

-normally masked by neuraminic acid
-neuraminidase (produced by all serotypes of S. pneumoniae)
cleaves n-acetyl neuraminic acid from cell surface, exposing TF antigen
-host in response produces IgM antibodies, which bind to the TF antigen
-Immune response cascade ensues, culminating in HUS (red cells, platelets,
glomerular endothelial cell damaged)
-TF antigen also found on hepatocytes
(explains hepatic dysfunction in some patients)

Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Atypical Hemolytic Uremic Syndrome:
Classification

A) Familial Form
-less than 20% of cases
-poor prognosis: end-stage renal disease or death of 50 to 80%
-First described in 1965: combination of hemolytic anemia & azotemia
in concordant monozygous twins.1
-Typically reported in children but infrequently, in adults
-Both autosomal dominant and recessive patterns of inheritance. 2

1. Campbell S, Carré IJ. Fatal haemolytic uraemic syndrome and idiopathic hyperlipaemia in monozygotic
twins. Arch Dis Child 1965;40:654-8.
2. Kaplan BS, Chesney RW, Drummond KN. Hemolytic uremic syndrome in families.N Engl J Med
1975;292:1090-3.
 Atypical Hemolytic Uremic Syndrome:
Classification(cont’d)
B) Sporadic Form
-Designation when no family history of the disease
-50% of cases appear to be idiopathic.
-Triggers:
--1) human immunodeficiency virus
--2) cancer
--3) organ transplantation
----de novo TMA reported in 3.6 to 14.0% of all kidney transplant recipients;
associated with humoral rejection & calcineurin inhibitors. 1-3
--4) pregnancy
---- ~10 to 15% of female patients with atypical HUS
develops during pregnancy or post partum. 4,5
--5) Medications:
----a) anticancer drugs
----b) immunotherapeutic agents (e.g., cyclosporine and tacrolimus)
----c) antiplatelet agents (e.g., ticlopidine and clopidogrel).

1. Ruggenenti P. Kidney Int 2002; 62:1093-104.

2. Karthikeyan V, Parasuraman R, Shah V, Vera E, Venkat KK. Am J Transplant 2003;3:1289-94.
3. Zarifian A, Meleg-Smith S, O’Donovan R, Tesi RJ, Batuman V. Kidney Int 1999;55:2457-66.
4. Noris M, Remuzzi 1. G. Hemolytic uremic syndrome. J Am Soc Nephrol 2005; 16:1035-50.
5. Besbas N, Karpman D, Landau D, et al. Kidney Int 2006;70:423-31.
6. Ruggenenti P, Noris M, Remuzzi G. Kidney Int 2001;60: 831-46.
7. Zakarija A, Bennett C. Semin Thromb Hemost 2005;31:681-90.
 Atypical HUS Association with
Complement System Abnormalities
-Since 1974, reduced serum levels of complement fraction C3 with normal
levels of C4 reported in patients with atypical HUS, reflecting complement
activation and consumption.1-3
-Genetic abnormalities in complement system proteins documented in both
familial and sporadic forms 4-11
-Two patients with Stx-related HUS reported with mutations in complement
regulatory genes 10,12
-Mutations in complement pathway have been found to account for 50 to 60%
of cases of atypical HUS13
1. Carreras L, Romero R, Requesens C, et al. JAMA 1981;245:602-4.
2. Stühlinger W, Kourilsky O, Kanfer A, Sraer JD. Lancet 1974;2:788-9.
3. Noris M, Ruggenenti P, Perna A, et al. J Am Soc Nephrol 1999;10:281-93.
4. Caprioli J, Noris M, Brioschi S, et al. Blood 2006;108:1267-79.
5. Noris M, Brioschi S, Caprioli J, et al. Lancet 2003;362:1542-7.
6. Richards A, Kemp EJ, Liszewski MK, et al. Proc Natl Acad Sci U S A 2003;100:12966-71.
7. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. Proc Natl Acad Sci U S A 2007;104:240-5.
8. Frémeaux-Bacchi V, Miller EC, Liszewski MK, et al. Blood 2008; 112:4948-52.
9. Le Quintrec M, Lionet A, Kamar N, et al. Am J Transplant 2008;8:1694-701.
10. Fang CJ, Fremeaux-Bacchi V, Liszewski MK, et al. Blood 2008;111:624-32.
11. Fakhouri F, Jablonski M, Lepercq J, et al. Blood 2008;112: 4542-5.
12. Loirat C, Noris M, Fremeaux-Bacchi V. Pediatr Nephrol 2008;23:1957-72.
13. Noris M, Remuzzi G. N Engl J Med. 2009;361:1676-87
 Overview of Complement System
in Hemolytic Uremic Syndrome
-Consists of plasma and membrane-bound proteins
that protect against invading organisms.1
-Composed of three activation pathways — classic, lectin, and alternative
-Pathways produce protease complexes termed C3 and C5 convertases
that cleave C3 and C5, leading to the membrane-attack complex
-C3 convertases of the classic and lectin pathways are formed by
C2 and C4 fragments
-Alternative pathway convertase cleaves C3 but not C4.1
-C3 hydrolysis in plasma initiates the alternative pathway, leading to the
deposition of C3b onto almost all plasma-exposed surfaces 2

1. Walport MJ. Complement: first of two parts. N Engl J Med 2001;344:1058-66.

2. Devaux P, Christiansen D, Fontaine M, Gerlier D. Control of C3b and C5b deposition by CD46 (membrane
cofactor protein) after alternative but not classical complement activation. Eur J Immunol 1999; 29:815-22.
Noris M, Remuzzi G. Hemolytic uremic syndrome. J Am Soc Nephrol. 2005 Apr;16(4):1035-50.
 Overview of Complement System
in Hemolytic Uremic Syndrome (cont’d)
-On host cells, membrane-anchored and fluid-phase regulators control complement activation :
----favoring the cleavage of C3b to inactive C3b (iC3b) by complement factor I (CFI) activity
----dissociating the multicomponent C3 and C5 convertases (i.e., decay-acceleration activity).
-Without normal regulation, C3b deposition:
----increases by more than a factor of 20 through the amplification loop 1
----causes activation of the complement cascade, which remains active until complement components
are consumed.
-Foreign targets and injured cells are attacked by complement due to:
----a) inability to bind soluble regulators
----b) no membrane-bound regulators .
-On the surface of bacteria, C3b binds to specific receptors on neutrophils and macrophages,
resulting in phagocytosis of complement-tagged bacteria.
-Therefore in a patient with atypical HUS, low serum C3 level with normal C4 level indicates selective
alternative pathway activation 2

1. Devaux P, Christiansen D, Fontaine M, Gerlier D. Control of C3b and C5b deposition by CD46 (membrane cofactor protein)
after alternative but not classical complement activation. Eur J Immunol 1999; 29:815-22.
2. Noris M, Ruggenenti P, Perna A, et al. Hypocomplementemia discloses genetic predisposition to hemolytic uremic syndrome
and thrombotic thrombocytopenic purpura: role of factor H abnormalities. J Am Soc Nephrol 1999;10:281-93.
Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular
thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular
thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Laboratory & Pathological Evidence of
Complement Abnormalities in Atypical HUS
-HUS patients w/low serum C3 levels have high levels of activated complement
components, including C3b, C3c, and C3d.1
-Granular C3 deposits in glomeruli & arterioles during acute disease
C/W activation of complement & local C3 consumption. 2,3
-C9 staining in glomeruli & small arteries with intimal proliferation and
thrombosis suggests activation up to the final lytic C5b-9 membrane-attack
complex.4

1. Kim Y, Miller K, Michael AF. Breakdown products of C3 and factor B in hemolytic-uremic syndrome. J Lab
Clin Med 1977;89:845-50.
2. Stühlinger W, Kourilsky O, Kanfer A, Sraer JD. Haemolytic-uraemic syndrome: evidence for intravascular
C3 activation. Lancet 1974;2:788-9.
3. Barré P, Kaplan BS, de Chadarévian JP, Drummond KN. Hemolytic uremic syndrome with
hypocomplementemia, serum C3NeF, and glomerular deposits of C3. Arch Pathol Lab Med 1977;101:357-61.
4. Landau D, Shalev H, Levy-Finer G, Polonsky A, Segev Y, Katchko L. Familial hemolytic uremic syndrome
associated with complement factor H deficiency. J Pediatr 2001;138:412-7.
 Complement factor H abnormalities
and Atypical HUS
-in 1981, Association of Familial Atypical HUS with Complement factor H abnormalities
first made in two brothers with atypical hemolytic–uremic syndrome who did not produce
complement factor H1
-The parents, who were first cousins, had half-normal CFH levels, indicating an inherited
defect.
-Complete or partial CFH deficiencies reported in patients with atypical HUS 2,3
-Reduced levels noted in asymptomatic family members of atypical HUS patients
-Clinical presentation from neonatal period to adulthood

1. Thompson RA, Winterborn MH. Hypocomplementaemia due to a genetic deficiency of beta 1H globulin. Clin Exp Immunol
1981;46:110-9.
2. Noris M, Ruggenenti P, Perna A, et al. Hypocomplementemia discloses genetic predisposition to hemolytic uremic syndrome
and thrombotic thrombocytopenic purpura: role of factor H abnormalities. J Am Soc Nephrol 1999;10:281-93.
3. Rougier N, Kazatchkine MD, Rougier JP, et al. Human complement factor H deficiency associated with hemolytic uremic
syndrome. J Am Soc Nephrol 1998;9:2318- 26.
 Complement factor H abnormalities
and Atypical HUS (cont'd)
-Mutations in the gene were first described in 19981
-Mutations account for 6–11% of cases of atypical HUS2
-factor H gene found on 1q32 alongside many other complement
regulatory genes1,3,4
-Most factor H mutations inherited in autosomal dominant fashion
w/variable penetrance

1. Warwicker P, Goodship TH, Donne RL, Pirson Y, Nicholls A, Ward RM, Turnpenny P, Goodship JA (1998) Kidney Int
53:836–844
2. Lee BH, Kwak SH, Shin JI, Lee SH, Choi HJ, Kang HG, Ha IS, Lee JS, Dragon-Durey MA, Choi Y, Cheong HI (2009)
Pediatr Res 66:336–340
3. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu S, Filler G, Pirson
Y, Wen LS, Atkinson JP, Goodship TH (2003) Proc Natl Acad Sci USA 100:12966–12971
4. Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P (2004) Mol
Immunol 41:355–367
 Factor H Autontibodies and Atypical HUS
-Factor H Autoantibodies account for 6 to 10% of patients with atypical HUS 1-3
-binding site localized to the Factor H C-terminus. 4
-inhibits the regulatory function of Factor H at cell surfaces by blocking its C-terminal recognition
region.
-mimics the effect of C-terminal FH mutations
-Dragon-Durey's Proposed Mechanism of Factor H Autoantibody Formation:
----affected children lack CFHR1 and CFHR3 secondary to homozygous deletions of the
corresponding genes.3,5,6
----Factor H Antibodies arise from an immune reaction against heterozygous mothers' CFHR1 and
CFHR3
----Evidence: Factor H Antibodies recognize CFHR1 and CFHR3 3
-Alternative explanation: CFHR1/3 deficiency may predispose to atypical HUS
(Patients with CFHR1/3 deficiency lacking anti-CFH antibodies develop atypical HUS. 6,7

1. Dragon-Durey MA, Loirat C, Cloarec S, et al. J Am Soc Nephrol 2005;16:555- 63.

2. Skerka C, Józsi M, Zipfel PF, Dragon-Durey MA, Fremeaux-Bacchi V. Thromb Haemost 2009;101:227-32.
3. Dragon-Durey MA, Blanc C, Marliot F, et al. J Med Genet 2009;46:447-50.
4. Józsi M, Strobel S, Dahse HM, et al. Blood 2007; 110:1516-8.
5. Józsi M, Licht C, Strobel S, et al. Blood 2008;111:1512-4.
6. Zipfel PF, Edey M, Heinen S, et al. PLoS Genet 2007;3(3):e41.
7. Koziolek MJ, Zipfel PF, Skerka C, et al. Kidney Int 2008;74:384-8.
 Complement factor I

-Serum-based member (fluid component)

of the complement regulation system (like Factor H)
-predominantly produced in the liver
-serine protease that cleaves C3b
(prevents formation of C3 convertase [C3bBb] from C3b)
-plays a key inhibitory role in preventing alternative pathway amplification1
-function depends on many co-factors,
(including factor H and C4-binding protein)
-gene found on chromosome 4

1. Noris M, Remuzzi G (2009) Atypical hemolytic-uremic syndrome. N Engl J Med 361:1676–1687

2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Complement factor I in HUS

-mutations causing HUS first described in 20041

-mutations found in sporadic cases rather than familial,
suggesting low penetrance of mutations
-abnormal gene encodes truncated factor I protein lacking
C-terminal serine proteinase region
-Alternative pathway dysregulation leads to TMA (to similar factor H deficiency),

with consumptive depletion of C3 and factor B

-Complete hereditary deficiency of factor I reported in > 30 pedigrees;
associated with severe pyogenic infections1

1. Fremeaux-Bacchi V, Dragon-Durey MA, Blouin J, Vigneau C, Kuypers D, Boudailliez B, Loirat C, Rondeau

E, Fridman WH (2004) Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome. J
Med Genet 41:e84
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Serum-Based Complement Regulators
(Factor H & Factor I)
-Synthesized in liver
-mutations or directed antibodies have poorer prognosis
-develop recurrence in their grafts
(therefore isolated renal transplantation not recommended at present)1
-isolated liver transplant or combined liver/kidney transplant
can be considered, but only in centres with previous experience1
-Factor H & I knockout mice w/uncontrolled alternative pathway activation
do not develop HUS; instead develop mesangial C3 deposits2

1. Taylor CM, Machin S, Wigmore SJ, Goodship TH (2010) Clinical practice guidelines for the management of
atypical haemolytic uraemic syndrome in the United Kingdom. Br J Haematol 148:37–47
2. Richards A, Kavanagh D (2009) Pathogenesis of thrombotic microangiopathy: insights from animal models.
Nephron Exp Nephrol 113:e97–e103
3. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Membrane co-factor protein
(MCP or CD46)
-Widely expressed transmembrane complement regulator
(expressed on almost every cell except erythrocytes)
-Works with factor I to degrade C3b and C4b
(which are bound to the host cell surface)
-Gene, like Factor H, found on chromosome 1q321

1. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu S,
Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship TH (2003) Mutations in human complement regulator,
membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc
Natl Acad Sci USA 100:12966–12971
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Membrane co-factor protein
(MCP or CD46) in HUS
-mutations cause incorrectly processing of Membrane co-factor protein;
therefore remains intracellular, affecting C3b binding
-Both autosomal dominant inheritance w/variable penetrance &
autosomal recessive patterns of inheritance
-In some pedigrees, carriers do not develop HUS
(mutation alone not always enough sufficient for disease development?)
-endothelial insult triggers overwhelming complement activation and TMA
-membrane-bound and not serum-bound, therefore renal transplantation
successful1

1. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu S,
Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship TH (2003) Mutations in human complement regulator,
membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc
Natl Acad Sci USA 100:12966–12971
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Thrombomodulin
-ubiquitous transmembrane endothelial cell glycoprotein with
anticoagulant, anti-inflammatory and cytoprotective properties1
-anchored to the cell by short cytoplasmic tail & single transmembrane domain
-In vitro, binds C3b and complement factor H
-negatively regulates complement by accelerating factor I-mediated inactivation
of C3b in the presence of co-factors (factor H and C4b binding protein)
-promotes activation of plasma procarboxypeptidase B (AKA: thrombin
activatable fibrinolysis inhibitor = TAFI)
-accelerates inactivation of anaphylatoxins C3a and C5a
-accelerates thrombin-mediated activation of protein C, down-regulating further
thrombin generation & suppressing clot formation.
-interferes with inflammation by suppressing leukocyte trafficking and
dampening complement activation.

1. Delvaeye M, Noris M, De Vriese A, Esmon CT, Esmon NL, Ferrell G, Del-Favero J, Plaisance S, Claes B,
Lambrechts D, ZojaC, Remuzzi G, Conway EM (2009) Thrombomodulin mutationsin atypical hemolytic-
uremic syndrome. N Engl J Med 361:345–357
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Thrombomodulin in HUS

-Mutations in lectin-like domain alter factor H and C3b binding & thus
complement regulation
-Mutations in the serine–threonine-rich region alter factor I-mediated
Cb3 inactivation
-Although solid phase protein, one patient w/thrombomodulin mutation
had recurrence of atypical HUS post-renal transplantation1;
Hmmmmm.....

1. Delvaeye M, Noris M, De Vriese A, Esmon CT, Esmon NL, Ferrell G, Del-Favero J, Plaisance S, Claes B,
Lambrechts D, ZojaC, Remuzzi G, Conway EM (2009) Thrombomodulin mutationsin atypical hemolytic-
uremic syndrome. N Engl J Med 361:345–357
2. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic
microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Complement Factor B (CFB) &
Complement fraction C3 in HUS
-Gain-of-function mutations can affect genes encoding these alternative pathway
C3 convertase components1,2
-CFB mutations, leading to chronic alternative-pathway activation,
occur in only 1-2% of Atypical HUS patients 1
-Mutant CFB’s have excess C3b affinity --> formation of hyperactive C3 convertase
resistant to dissociation, enhancing C3b formation [17]
-Heterozygous mutations in C3 occur in ~ 4 to 10% of Atypical HUS patients,
usually with low C3 levels2
-Most C3 mutations reduce C3b binding to CFH and MCP,
severely impairing degradation of mutant C3b 2

1. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, et al. Gain-of-function mutations in complement

factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci U S A 2007;104:240-5.
[Erratum, Proc Natl Acad Sci USA 2007;104:10749.]
2. Frémeaux-Bacchi V, Miller EC, Liszewski MK, et al. Mutations in complement C3 predispose to
development of atypical hemolytic uremic syndrome. Blood 2008; 112:4948-52
3. Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009 Oct 22;361(17):1676-87.
Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009 Oct 22;361(17):1676-87.
Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009 Oct 22;361(17):1676-87.
Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009 Oct 22;361(17):1676-87.
Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009 Oct 22;361(17):1676-87.
 Vascular endothelial growth factor (VEGF)
-most important endothelial growth factor; vital in maintaining healthy, normally
functioning endothelial cells1
-Produced in the kidney by the podocyte
-In adults, monoclonal antibodies against VEGF-A (bevacizumab) used in some
cancer therapy regimens (VEGF is found to be upregulated in many human
tumours); subset with renal side effects during treatment2
-Manifestations of renal injury: from proteinuria and hypertension to
glomerular TMA & AKI
-Investigation of VEGF in Shiga-toxin-induced HUS3:
----Recent evidence stx modulates VEGF-A production of human, but not
murine origin
----In vitro, human podocytes exposed to stx produce 60% less VEGF-A
compared with controls
1. Eremina V, Baelde HJ, Quaggin SE (2007) Role of the VEGF—a signaling pathway in the glomerulus: evidence for crosstalk
between components of the glomerular filtration barrier. Nephron Physiol 106:p32–p37
2. Izzedine H, Massard C, Spano JP, Goldwasser F, Khayat D, Soria JC (2010) VEGF signalling inhibition-induced proteinuria:
mechanisms, significance and management. Eur J Cancer 46:439–448
3. Psotka MA, Obata F, Kolling GL, Gross LK, Saleem MA, Satchell SC, Mathieson PW, Obrig TG (2009) Shiga toxin 2 targets
the murine renal collecting duct epithelium. Infect Immun 77:959–969
4. Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular thrombotic microangiopathy. Pediatr
Nephrol. 2011 Apr;26(4):523-33
Ariceta G, Besbas N, Johnson S, Karpman D, Landau D, Licht C, Loirat C, Pecoraro C, Taylor CM, Van de Kar N,
Vandewalle J, Zimmerhackl LB; European Paediatric Study Group for HUS. Guideline for the investigation and initial therapy
of diarrhea-negative hemolytic uremic syndrome. Pediatr Nephrol. 2009 Apr;24(4):687-96.
Ariceta G, Besbas N, Johnson S, Karpman D, Landau D, Licht C, Loirat C, Pecoraro C, Taylor CM, Van de Kar N,
Vandewalle J, Zimmerhackl LB; European Paediatric Study Group for HUS. Guideline for the investigation and initial therapy
of diarrhea-negative hemolytic uremic syndrome. Pediatr Nephrol. 2009 Apr;24(4):687-96.
Ariceta G, Besbas N, Johnson S, Karpman D, Landau D, Licht C, Loirat C, Pecoraro C, Taylor CM, Van de Kar N,
Vandewalle J, Zimmerhackl LB; European Paediatric Study Group for HUS. Guideline for the investigation and initial therapy
of diarrhea-negative hemolytic uremic syndrome. Pediatr Nephrol. 2009 Apr;24(4):687-96.
 Exceptions to guideline
(a) When effect of an alternative treatment anticipated. (Ex. HUS in a sibling of a
patient with congenital ADAMTS13 deficiency likely to have same diagnosis ;
might be expected to respond to plasma infusion 10 ml/kg per day.
(b) Clinical presentation strongly suggestive of early onset cobalamin-C disorder
(feeding difficulty, failure to thrive, hypotonia, lethargy, leukopenia and
megaloblastic anemia).
(c) Technical difficulties in achieving vascular access in small children.
(d) Risks of plasmapheresis outweigh the benefits in a child with apparently mild renal
involvement and conserved urine output

Ariceta G, Besbas N, Johnson S, Karpman D, Landau D, Licht C, Loirat C, Pecoraro C, Taylor CM, Van de Kar N,
Vandewalle J, Zimmerhackl LB; European Paediatric Study Group for HUS. Guideline for the investigation and initial therapy
of diarrhea-negative hemolytic uremic syndrome. Pediatr Nephrol. 2009 Apr;24(4):687-96.
European Paediatric Study Group for HUS. Guideline for the investigation and initial therapy of diarrhea-negative hemolytic
uremic syndrome. Pediatr Nephrol. 2009 Apr;24(4):687-96.
Ruggenenti P, Noris M, Remuzzi G (2001) Thrombotic microangiopathy, hemolytic uremic syndrome,
and thrombotic thrombocytopenic purpura. Kidney Int 60:831-46.
 Eculizumab
-monoclonal antibody that blocks complement activity via C5 cleavage
-Case reports of treatment of atypical HUS or preventing relapse after
transplantation [48-53] (demonstrate possible effectiveness of inhibiting
complement activation in atypical HUS)
-ongoing "Clinical trial of eculizumab in pediatric atypical HUS"
investigating role in the treatment of Atypical HUS due to
complement dysregulation.

48. Gruppo RA, Rother RP. Eculizumab for congenital atypical hemolytic-uremic syndrome. N Engl J Med 2009; 360:544.
49. Nürnberger J, Philipp T, Witzke O, et al. Eculizumab for atypical hemolytic-uremic syndrome. N Engl J Med 2009; 360:542.
50. Zimmerhackl LB, Hofer J, Cortina G, et al. Prophylactic eculizumab after renal transplantation in atypical hemolytic-uremic
syndrome. N Engl J Med 2010; 362:1746.
51. Lapeyraque AL, Frémeaux-Bacchi V, Robitaille P. Efficacy of eculizumab in a patient with factor-H-associated atypical
hemolytic uremic syndrome. Pediatr Nephrol 2011; 26:621.
52. Prescott HC, Wu HM, Cataland SR, Baiocchi RA. Eculizumab therapy in an adult with plasma exchange-refractory atypical
hemolytic uremic syndrome. Am J Hematol 2010; 85:976.
53. Al-Akash SI, Almond PS, Savell VH Jr, et al. Eculizumab induces long-term remission in recurrent post-transplant HUS
associated with C3 gene mutation. Pediatr Nephrol 2011; 26:613.
 Eculizumab dosing (via manufacturer
as per Up to Date)
Induction dosing Maintenance dosing
(based on patient body weight) (based on patient body weight)

5 to <10 kg – 300 mg, one weekly dose 5 to <10 kg – 300 mg every three weeks,
10 to <20 kg – 600 mg, one weekly dose starting week 2
20 to <30 kg – 600 mg, two weekly doses 10 to <20 kg – 300 mg every two weeks,
30 to <40 kg – 600 mg, two weekly doses starting week 2
20 to <30 kg – 600 mg every two weeks,
≥40 kg – 900 mg, four weekly doses
starting week 3
30 to <40 kg – 900 mg every two weeks,
starting week 3
≥40 kg – 900 mg, 1200 mg every two
weeks, starting week 5

*Of note, the doses for young children were calculated from a pharmacokinetic model derived from adult data because there are
no data in children. In some children, the above dosing protocol has not completely blocked complement activation, and in these
patients, dosing of eculizumab has been increased. If eculizumab is used in young children, complement activity should be
assessed by measuring CH50
Unanswered Questions
 Pop Quiz

Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular

thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Pop Quiz

Keir L, Coward RJ. Advances in our understanding of the pathogenesis of glomerular

thrombotic microangiopathy. Pediatr Nephrol. 2011 Apr;26(4):523-33
 Cincinnati Children’s Nephrology Tests
http://www.cincinnatichildrens.org/svc/alpha/n/nephrology-hypertension/services/tests/comp-profiles.htm

Complete Complement Profile (Comp Prof)

Note: Tests can be performed individually, or in a Profile package.
C1Q C5 C9 Properdin
C2 C6 Factor H C4 Binding Protein
C3 C7 Factor I C1 Inhibitor
C4 C8 Factor B Nephritic Factor (optional)
This profile is useful in the detection of inherited complement deficiencies which may predispose to lupus-
like syndromes, to recurrent bacterial infection, particularly Neisseria, or to hereditary angioedema. In
association with the measurement of nephritic factor, this profile aids in distinguishing acute from chronic
nephritis and in identifying the three types of membranoproliferative glomerulonephritis. A nephrologist
interprets all Complete Complement Profiles.
Specimen requirements 1 mL Serum, frozen.

Nutritional Complement Profile (NUTRIT PRO)

Factor B Factor H Factor I C3
For distinguishing low levels of C3 resulting from undernutrition from those due to complement activation. A
nephrologist interprets all Nutritional Complement Profiles.
Specimen requirements 1 mL Serum, frozen.

C3 Nephritic Factor (C3 NEF)

An autoantibody that depresses the serum level of C3 and, secondarily, other complement components.
Among the hypocomplementemic glomerulonephritides, nephritic factor is absent in acute post-
streptococcal glomerulonephritis and lupus nephritis. It is present in most hypocomplementemic patients
with membranoproliferative glomerulonephritis and also those with partial lipodystrophy. Although the
antibody may be instrumental in producing the nephritis, its level is not closely associated with the clinical
course.
Specimen requirements 1 mL Serum, frozen.
 Contact the Nephrology Clinical Laboratory at Cincinnati Children's
http://www.cincinnatichildrens.org/svc/alpha/n/nephrology-
hypertension/services/contact.htm
Phone Mailing Address
Clinical Lab 513-636-4530 Children's Hospital Medical Center
Clinical Lab Results 513-636-4281 Division of Nephrology and Hypertension
MLC 7022
Clinical Lab Processing 513-636-7355
3333 Burnet Avenue
Pager 513-736-4617 Cincinnati, Ohio 45229-3039
Fax 513-636-8924
Mailing Address-Specimens
E-mail Children's Hospital Medical Center
Prasad Devarajan: Lab Processing B-4
Prasad.Devarajan@cchmc.org 3333 Burnet Avenue
Thelma Kathman:Thelma.Kathman@cchmc.org Cincinnati, Ohio 45229-3039
 University of Iowa Molecular Otolaryngology & Renal Research Laboratory
http://www.healthcare.uiowa.edu/labs/morl/FH%20Autoantibodies.htm

Factor H Autoantibodies
The Clinical Diagnostics Service of the Molecular Otolaryngology Research Laboratory is a Joint Commission-approved CLIA-
accredited diagnostic laboratory.

Factor H Autoantibodies
Dense Deposit Disease (DDD, aka Membranoproliferative Glomerulonephritis Type II, MPGNII)
Factor H autoantibodies have been associated with DDD (Meri et al., 1992). In patients with DDD, these autoantibodies bind to
and block the N-terminal region of the Factor H protein, which compromises its fluid-phase regulatory function.

Atypical Hemolytic-Uremic Syndrome

Factor H autoantibodies are identified in ~10% patients with aHUS (Dragon-Durey, et al 2005, Moore, et al 2010). Most but not
all patients with aHUS who develop Factor H autoantibodies are homozygous for a known polymorphism, del(CFHR3-CFHR1).
Homozygosity for this deletion is seen in 15% of patients with aHUS as compared to 5% of controls of northern European
ancestry (Zipfel et al 2007, Skerka et al 2009). The Factor H autoantibodies in aHUS patients bind to and block the C-terminal
region of the Factor H protein, which interferes with its surface regulatory function (Józsi et al 2007).

MORL screening methodology

Enzyme Linked Immuno-Sorbent Assay (ELISA) is used to assay the presence of Factor H autoantibodies (Dragon-Durey, et
al 2005, Józsi et al 2007, Moore, et al 2010).

Sensitivity
>99%

Turn-around time
Turn around time is approximately 1 month.

Cost: $324

Indications for screening

Screening is appropriate for patients with aHUS and biopsy-proven DDD..
 University of Iowa Molecular Otolaryngology & Renal Research Laboratory
http://www.healthcare.uiowa.edu/labs/morl/FH%20Autoantibodies.htm

The Clinical Diagnostics Division of the Molecular Otolaryngology Research Laboratories is a Joint Commission-approved
CLIA-accredited diagnostic laboratory that offers mutation screening of several genes that have been implicated in a few rare
kidney diseases.

C3 encodes the protein Complement Component 3. C3 is an acute-phase reactant and is up-regulated during acute
inflammation. Mutations in C3 have been found in persons with atypical Hemolytic Uremic Syndrome (aHUS).

Factor H (CFH or HF1) encodes the protein complement FACTOR H (HF1), a member of the alternative pathway of the
complement cascade. Mutations in CFH have been found in persons with atypical Hemolytic Uremic Syndrome (aHUS) and
Dense Deposit Disease (DDD, also known as Membranoproliferative Glomerulonephritis Type II or MPGNII). Screening is
offered to persons with aHUS and biopsy-proven DDD; we also offer screening for some other factor H-related diseases.

Factor I (CFI) encodes the protein complement FACTOR I (CFI), a proteolytic enzyme that destroys the hemolytic and
immune-adherence activities of cell-bound, activated C3. Mutations in FI have been found in persons with atypical Hemolytic
Uremic Syndrome (aHUS).

Membrane Cofactor Protein (MCP) encodes the protein Membrane Cofactor Protein (MCP, CD46), a locally synthesized
membrane bound complement regulator. Mutations in MCP have been found in persons with atypical Hemolytic Uremic
Syndrome (aHUS).

Factor B (CFB) is a zymogen that carries the catalytic site of the complement alternative pathway convertase C3bBb. The
FB gene contains 18 exons and encodes the 764 amino acid protein, Complement Factor B. There have been two patients
reported with gain-of-function mutations in Factor B.

Factor H Related 5 is a member of the Factor H-Related gene family. It has C-reactive protein and heparin binding
properties. Several single nucleotide polymorphisms in CFHR5 have been associated in persons with biopsy-proven DDD.

Testing turn-around time is approximately three months.