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Familial hemolytic uremic syndrome associated with complement factor H deficiency
CLINICAL AND LABORATORY OBSERVATIONS
Familial hemolytic uremic syndrome associated with complement factor H deficiency Daniel Landau, MD, Hannah Shalev, MD, Gal Levy-Finer, MD, Alexandra Polonsky, MD, Yael Segev, PhD, and Leonid Katchko, MD Atypical hemolytic uremic syndrome (HUS) associated with factor H deficiency (FHD) carries a poor prognosis. A 3-year-old girl with FHD-HUS reached end-stage renal disease at age 6 months after experiencing numerous relapses; she underwent a cadaveric renal transplant at age 46 months. One month after transplantation, she experienced an extensive non-hemorrhagic cerebral infarction. Later, hematologic and renal manifestations of HUS developed, followed by another massive cerebral infarction and death in spite of multiple plasma transfusions. A 14-month-old boy with FHDHUS experienced numerous HUS episodes starting at the age of 2 weeks. Daily plasma transfusions during relapses brought about only a temporary state of remission. However, prophylactic twice-weekly plasma therapy has been successful in preventing relapses and preserving renal function. With this regimen, serum factor H was increased from 6 mg/dL to subnormal values of 12 to 25 mg/dL (normal >60 mg/dL). We conclude that FHD-HUS recurs because FHD is not corrected by renal transplantation. A hypertransfusion protocol may prevent FHD-HUS. (J Pediatr 2001;138:412-7)
Atypical, diarrhea-negative forms of hemolytic uremic syndrome are a heterogeneous group of disorders. Inherited forms of HUS should be suspectFrom the Departments of Pediatrics and Pathology, Soroka University Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Submitted for publication May 26, 2000; revision received Sept 11, 2000; accepted Oct 26, 2000. Reprint requests: Daniel Landau, MD, Pediatric Nephrology, Department of Pediatrics “B,” Soroka Medical Center, PO Box 151, Beer Sheva 84101, Israel. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/112649 doi:10.1067/mpd.2001.112649
412
ed in any case of atypical HUS occurring among more than one family member on a non-epidemic basis.1 Some of these inherited forms of HUS have been associated with abnormalities in the complement system. Complement factor H, a 150-kd regulatory
See editorial, p 303. molecule of the complement cascade, plays a central role in the inhibition of the ongoing complement consumption in response to different stimuli.2 Recently, factor H deficiency has been implicated in the pathogenesis of atypical HUS.3,4 We have recently described an extended Bedouin family
with an autosomal recessive type of HUS, associated with severe FHD.5 This deficiency is due to a defective post-translational processing of a mutant factor H gene and is associated with complement consumption.6 Disease onset has been as early as 2 weeks of age, and the outcome has been very poor: all children have either died or reached end-stage renal disease by the end of the first year of life. No intervention, such as plasma infusion, plasma exchange, or intravenous immune globulin, has provided long-term efficacy in previous cases. In addition, no clear data exist regarding the risk for recurrence of this disease after renal transplantation. We report a patient with familial FHD-HUS who developed serious HUS after renal transplantation. In another patient, the efficacy of a novel therapy is presented. CNS ESRD FFP FHD HUS
Central nervous system End-stage renal disease Fresh frozen plasma Factor H deficiency Hemolytic uremic syndrome
METHODS Factor H measurement Serum samples were separated from whole blood and kept at –20°C until they were examined. Serum factor H levels were determined by using a radial immunodiffusion commercial kit (The Binding Site Co, Birmingham, UK), based on polyclonal antibodies in
LANDAU ET AL
THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 3 an agarose gel. The kit contains 3 calibrator solutions with predefined factor H concentrations (7, 42, and 70 mg/dL), to be used as internal controls. Five microliters of serum was applied to the agar plates and incubated for 72 hours in room temperature and controlled humidity conditions. The obtained rings’ radiuses were measured with a radial immunodiffusion plate reader (Grafar Co, Rockwell, Md) and compared with a reference table to determine factor H concentrations. All sample readings were done in duplicate.
Immunohistochemistry Immunohistochemical staining was carried out by the avidin-biotin-peroxidase complex method as previously described.7 An anti-human C5b-9 monoclonal antibody was used (Metra Biosystems, Mountain View, Calif). Deparaffinized rehydrated sections were incubated with nonimmune horse serum, followed by incubation with the monoclonal primary antibody (5 µg/mL) and biotin-labeled horse antimouse immunoglobulin. Then, an avidin-biotin-peroxidase complex was added, and this was visualized by addition of chromogen (DAB-H2O2 solution), producing a brown color of the reaction product. The sections were counterstained with hematoxylin. Negative control kidney biopsy slides (from a patient with mild proteinuria, with no histologic changes as determined by light microscopy and immunofluorescence) were treated similarly.
A
B
Case Descriptions
Fig 1. A, Electron microscopic (original magnification ×4500) photograph of allograft kidney biopsy specimen from patient 1, obtained on post-transplantation day 30, consistent with thrombotic microangiopathy. Glomerular capillary loop showing 2 swollen endothelial cells (asterisk) detached from the basement membrane.The subendothelial space shows fibrin deposition and platelet aggregation (open arrow).There is no evidence of immune complexes around the capillary basement membrane. Epithelial cell podocytes are effaced. B, Immunohistochemical staining of the allograft kidney biopsy specimen for C5b-9 (terminal attack complex). A part of the glomerulus is shown (original magnification ×800), with intracapillary fibrin thrombi (black arrows). C5b-9 immunostains positively in capillary walls (open arrows) and mesangium (asterisk).
CASE 1. A 46-month-old Bedouin girl was found at birth to have low serum C3 levels (250 mg/L). She was the fourth child of consanguineous parents, members of an extended Bedouin family with hypocomplementemic HUS associated with FHD, previously described.5 Her two younger sisters died of familial HUS at the ages of 2 weeks and 3 months. Complete blood count and serum urea and creatinine levels were normal after birth. At age 3
weeks, she was admitted to the hospital for the first time because of vomiting and lethargy. Microangiopathic hemolytic anemia (plasma hemoglobin of 8.4 g/100 mL, schistocytes, reticulocytosis, thrombocytopenia (102,000) and renal insufficiency (increases in creatinine and urea nitrogen values to 114.9 µmol/L [1.3 mg/100 mL] and 8.56 mmol/L [24 mg/100 mL], respectively)
were observed. Hypocomplementemia (C3: 370 mg/L) persisted. Serum factor H levels were very low (<6 mg/100 mL; normal >60).8 Findings on a renal biopsy specimen obtained at age 3 weeks were consistent with HUS, as previously described.5 She was treated with daily doses of fresh frozen plasma (10 mL/kg) for 3 days, which caused a transient clinical improvement, be413
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Fig 2. Brain computed tomography of patient 1 shows extensive parietal infarctions on the distribution of both middle cerebral arteries. In addition, an area of right subacute ischemia with “luxury perfusion” (arrow), as well as a left subacute ischemia with basal ganglia involvement, is seen. An old left posterior cerebral infarction on the distribution of the posterior cerebral artery (arrowhead) is also seen.
cause shortly thereafter she developed recurrent HUS relapses (none of which were associated with central nervous system involvement). No other specific therapy was provided. At age 6 months, peritoneal dialysis was initiated for anuric ESRD. She did not experience further HUS relapses as ESRD developed. Serum C3 levels during ESRD remained low. At age 44 months, she underwent cadaveric renal transplantation. The postoperative course was uneventful: urine output was adequate, and serum creatinine concentration decreased from 419.9 µmol/L (4.8 mg/100 mL) to 19.4 µmol/L (.2 mg/100 mL) within days. The patient received tacrolimus, azathioprine, and prednisone. In addition, she received low-dose aspirin, ranitidine, and prophylactic co-trimoxazole. Her blood pressure was well controlled with atenolol, minoxidil, and clonidine. On post-transplantation day 16, a left hemiparesis was noted. Her blood 414
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Fig 3. Patient 2: serum creatinine (1000*mg/dL) (open squares) and LDH (U/L) (black circles) levels since birth. Blood transfusions (arrows) and elevation of serum creatinine and LDH levels reflect a state of HUS relapse. LDH, Lactate dehydrogenase; FFP PRN, fresh frozen plasma (10 mL/kg/dose) given daily during relapses; IFN-γ, interferon-γ (2.5 U/kg/dose, 3 times a week); FFP qw, FFP (15 mL/kg/dose) given as prophylaxis once a week; FFP BIW, FFP (15-20 mL/kg/dose) given as prophylaxis twice a week. pressure was well controlled (110/70 mm Hg). A large ischemic cerebral infarct extending along the right middle cerebral artery was demonstrated on brain computerized tomography. A workup for stroke predisposing conditions included search for bleeding tendency and/or thrombophilic states, none of which were present. Echocardiography demonstrated left ventricular hypertrophy and moderate mitral regurgitation without evidence of atrial thrombi. Two weeks later (1 month after transplantation), a deterioration in renal function was noted. Serum creatinine and urea concentrations increased from 17.6 µmol/L (.2 mg/100 mL) and 5.7 mmol/L (16 mg/100 mL) to 83 µmol/L (.94 mg/100 mL) and 27.1 mmol/L (76 mg/100 mL), respectively. A microangiopathic anemia picture was evident, and hypertension developed. A percutaneous kidney biopsy specimen revealed a histologic picture consistent with HUS relapse (Fig 1, A): fibrin thrombi filled the glomerular capillary lumina, and there was a segmental collapse of glomerular membranes. Electron microscopy
showed focal necrosis of endothelial cells and fibrin thrombi in glomerular capillary lumina but no evidence of immune complex deposition. No glomeruli were available for immunofluorescent investigation. Immunohistochemistry with a C5b-9 monoclonal antibody showed specific staining for this protein in glomerular capillary loops and mesangium (Fig 1, B). This staining was absent in a kidney biopsy specimen from a patient with minimal change disease. Mild evidence of tubulointerstitial rejection was also seen, with infiltration of mononuclear cells inside the venular walls. The child became stuporous, tachypneic, and pale. Repeat brain computed tomography (Fig 2) showed a new finding in the left cerebral hemisphere with the same diffuse low-density appearance previously noted on the right, consistent with a second ischemic cerebral infarct. A therapeutic trial with FFP did not prove effective. Neurologic examination showed evidence of brain death, and she died 3 days later, 34 days after transplantation. The family did not permit a postmortem examination.
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THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 3 CASE 2. A term male infant was found at birth to have low C3 levels (220 mg/L). Four previous siblings died of HUS and were previously reported.5 His serum creatinine and hemoglobin values were normal at birth, but at age 10 days, he had a first episode of HUS, manifested as renal failure (creatinine, 274 µmol/L, 3.1 mg/100 mL), hypertension, and elevated lactate dehydrogenase levels. He was treated with daily transfusions of FFP, 10 mL/kg, until signs of remission developed (Fig 3). However, he later experienced numerous relapses of the disease, with abnormal levels of serum creatinine, uric acid, lactate dehydrogenase, and triglycerides (increased during relapse), and hemoglobin (decreased during relapse). Blood pressure remained elevated, and left ventricular hypertrophy was evident at age 7 months. He required 16 blood transfusions over the first 8 months of life for repeated episodes of anemia (hemoglobin <1 g/100 mL). Serum factor H levels were very low (<6 mg/100 mL, normal >60). A trial with subcutaneous interferon-γ (2.5 U/kg/dose, three times a week), reported to increase factor H synthesis by hepatocytes,9 was given for 2 months. Factor H levels were only mildly elevated from baseline (8.2 mg/100 mL), but no long-term remission was achieved. A subsequent trial of weekly prophylactic FFP transfusion (10 mL/kg/dose) was also unsuccessful in preventing further relapses. At age 8.5 months, a twice-a-week regimen of FFP, 15 to 20 mL/kg/dose (from a single-donor pheresis source), administered through an implanted central venous catheter, was initiated. This treatment brought about a persistent decrease in serum creatinine concentration (26.5-39.8 µmol/L, 0.3-0.45 mg/100 mL) and a lower demand for blood transfusions (only one in the 7 months since the beginning of this intervention) (Fig 3). Blood pressure was better controlled, and left ventricular hypertrophy gradually regressed.
Fig 4. Serum factor H levels of patient 2 before any regular intervention (“zero”) and timely during a twice-weekly (Mondays and Thursdays) 20 mL/kg FFP protocol: 0.5, 24, 48, and 72 hours after finishing the transfusion.Three columns at right show factor H levels from patient’s (heterozygote) mother, healthy sibling, and infused FFP.
Factor H peak level after FFP administration was as high as 23 mg/100 mL. At trough, 72 hours after FFP administration, it was above 10 mg/100 mL. Factor H level in the transfused plasma sample was 61 mg/100 mL (Fig 4). Serum C3 levels remained low and did not change. At present, 9 months after the initiation of this treatment, there is no apparent increase in plasma requirements. Urinalysis shows no protein or blood.
DISCUSSION These cases add to our knowledge about the course of this unique form of atypical HUS. No further HUS relapses were encountered as soon as patient 1 progressed to ESRD. A similar long-term HUS remission was previously observed.5 Nevertheless, persistent hypocomplementemia indicated that the basic complement consumption process was ongoing.6 Even
though FHD may cause ongoing complement consumption, the causative role of complement consumption in HUS is not understood. The endothelium is the principal target tissue in HUS, and endothelial cells express factor H.10 The permissive role of the kidney endothelial bed in the pathogenesis of HUS has been previously shown in a group of adult patients with refractory HUS in whom remission was achieved only after bilateral nephrectomy.11 The fact that this atypical HUS variant “needs” functioning kidney tissue for its propagation is well portrayed in case 1: HUS recurred as soon as new functioning kidney tissue was introduced. Plasma obtained from patients with thrombotic thrombocytopenic purpura and patients with atypical HUS induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) only in small vessel dermal, renal, and cerebral lineages but not in pulmonary or hepatic lineages of microvascular endothelial 415
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cells.12 This dichotomy parallels the current clinical observation and supports the existence of organ-specific endothelial cells with different susceptibilities to injury. However, further evidence is needed to prove this hypothesis. FHD13 has been associated with several diseases, such as systemic lupus erythematosus,14 type II membranoproliferative glomerulonephritis,15 a form of chronic collagen type III glomerulopathy,16 and atypical familial HUS of both recessive5 and dominant17 inheritance. We have previously shown that this specific FHD-HUS is associated with very low serum factor H levels and is caused by a mutation in the factor H gene on chromosome 1. The defective protein product of the mutated factor H gene can be detected intracellularly but is not secreted by cultured fibroblasts.6 Factor H– deficient Norwegian pigs develop ESRD with pathologic characteristics of membranoproliferative glomerulonephritis.18 These animals have congenital FHD, associated with hypocomplementemia early in life and subsequent death from renal failure caused by massive complement deposition in glomeruli. Although no immune complex disease developed in our patients, C5b-9 was present in capillary loops in the child with post-transplantation HUS. No such immunostaining could be detected in normal kidney tissue. Complement activation on platelet-endothelial interaction is mediated by C5b-9 production on the endothelial surface, associated with a shift toward a more procoagulant state. In addition, it is now known that the response to C5b-9 injury is not merely due to the creation of holes in the cell membrane but may also be due to a “sublytic” effect.19 This phenomenon may have important influence in different renal diseases.20 However, further studies are needed to determine whether this C5b-9 staining in the damaged capillaries in our case is a specific phenomenon for FHD-HUS 416
THE JOURNAL OF PEDIATRICS MARCH 2001 or merely reflects a complement-mediated injury to endothelium. Overall, the absence of this circulating factor leads to kidney endothelial damage and ESRD. Renal transplantation does not correct this extrarenal deficiency, and therefore HUS relapses after this intervention. CNS involvement in HUS has been described for both the typical21,22 and atypical forms23 in up to 30% of cases. CNS involvement as a preliminary sign of post-transplantation HUS relapse has been previously reported.24 This was also a young patient with atypical HUS who did not exhibit any CNS involvement during the HUS episode that led to ESRD. HUS relapse in patients who have undergone renal transplantation has been previously described (mainly the atypical forms).25,26 CNS involvement has not occurred in the previous 11 children with primary FHD-HUS who have been treated (but have not undergone transplantation) and died in our institution in the past 20 years.5 Perhaps the severe CNS involvement was related to extensive endothelial damage, occurring when a large adult kidney was transplanted into a small child. This may have allowed the accumulation of abnormal von Willebrand factor multimers, which facilitates the deposition of platelet microthrombi.27 No therapy proved effective in the 11 patients that we treated in the past 20 years, including transfusions of FFP during relapses, intravenous immune globulin, or plasmapheresis. This is at variance with other reports28,29 that claim a therapeutic benefit of plasma therapy in atypical HUS. Plasmapheresis was effective in inducing remission and extending renal survival.30 However, the number of relapses was even higher, again suggesting that a circulating factor is only temporarily replaced (or removed) by plasma therapy (or exchange). Case 2 shows that FHD-HUS can be prevented by frequent (twice weekly) plasma therapy, when a partial correction of serum fac-
tor H levels occurs. The twice-a-week FFP transfusion protocol was adopted based on the experience with factor H–deficient Norwegian pigs. When these piglets were given weekly plasma transfusions, their survival was extended.31 In our patient, the plasma hypertransfusion protocol was able to increase serum factor H levels to only subnormal values, yet it was sufficient to prevent clinical relapses. C3 levels remain depressed with this protocol, suggesting that complement consumption continues. In addition, renal biopsy specimens from patients with FHDHUS do not show evidence of massive complement deposition,5 contrary to the porcine FHD model. Thus FFP transfusions, which increase serum factor H levels, are beneficial in preventing HUS relapses in spite of persistent low C3 levels, suggesting again a possible independent, protective role for factor H, irrespective of ongoing complement activation. Based on this new observation, in future cases of patients with FHD reaching ESRD that requires renal transplantation, preemptive and posttransplantation factor H replacement should be considered. This can currently be done by repeated plasma transfusions, to be started together with the grafting surgical procedure.
REFERENCES 1. Kaplan BS, Chesney RW, Drummond KN. Hemolytic uremic syndrome in families. N Engl J Med 1975;292: 1090-3. 2. Vik D, Munoz-Canovez P, Chaplin D, Tack B. Factor H. Curr Top Microbiol Immunol 1989;153:147-62. 3. Pichette V, Querin S, Schurch W, Brun G, Lehner-Netsch G, Delage JM. Familial hemolytic uremic syndrome and homozygous factor H deficiency. Am J Kidney Dis 1994;24:936-41. 4. Warwicker P, Goodship THJ, Donne RL, Pirson Y, Nicholls A, Ward RM, et al. Genetic studies into inherited and sporadic hemolytic uremic syndrome. Kidney Int 1998;53:836-44. 5. Ohali M, Shalev H, Schlezinger M, Katz Y, Katchko L, Carmi R, et al. Au-
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tosomal recessive hemolytic uremic syndrome associated with hypocomplementemia and decreased factor H. Pediatr Nephrol 1998;12:619-24. Ying L, Katz Y, Haider N, Schlesinger M, Carmi R, Shalev H, et al. Complement factor H gene mutation associated with autosomal recessive hemolytic uremic syndrome. Am J Hum Genet 1999;65:1538-46. Hsu SM, Raine L, Fanger H. Use of avidin-peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-80. Wolach B, Carmi D, Gilboa S, Satar M, Segal S, Dolfin T, et al. Some aspects of the humoral immunity and the phagocytic function in newborn infants. Isr J Med Sci 1994;30:331-5. Luo W, Vik DP. Regulation of complement factor H in a human liver cell line by interferon-gamma. Scand J Immunol 1999;49:487-94. Vastag M, Skopal J, Kramer J, Kolev K, Voko Z, Csonka E, et al. Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4. Immunobiology 1998;199:5-13. Remuzzi G, Galbusera M, Salvadori M, Rizzoni G, Paris S, Ruggeneti P. Bilateral nephrectomy stopped disease progression in plasma-resistant hemolytic uremic syndrome with neurological signs and coma. Kidney Int 1996;49:282-6. Mitra D, Jaffe EA, Weksler B, Hajjar KA, Soderland C, Laurence J. Thrombotic thrombocytopenic purpura and sporadic hemolytic-uremic syndrome plasmas induce apoptosis in restricted lineages of human microvascular endothelial cells. Blood 1997;89:1224-34. Jokiranta TS, Zipfel PF, Hakulinen J, Kuhn S, Pangburn MK, Tamerius JD, et al. Analysis of the recognition mechanism of the alternative pathway of com-
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plement by monoclonal anti-factor H antibodies: evidence for multiple interactions between H and surface bound C3b. FEBS Lett 1996;393:297-302. Fijen CA, Kuijper EJ, Te Bulte M, van de Heuvel MM, Holdrinet AC, Sim RB, et al. Heterozygous and homozygous factor H deficiency states in a Dutch family. Clin Exp Immunol 1996; 105:511-6. Jansen JH, Hogasen K, Grondahl AM. Porcine membranoproliferative glomerulonephritis type II: an autosomal recessive deficiency of factor H. Vet Rec 1995;137:240-4. Vogt VA, Wyatt RJ, Burke BA, Simonton SC, Kashtan CE. Inherited factor H deficiency and collagen type III glomerulopathy. Pediatr Nephrol 1995;9:11-5. Warwicker P, Donne RL, Goodship JA, Goodship TH, Howie AJ, Kumararatne DS, et al. Familial relapsing haemolytic uraemic syndrome and complement factor H deficiency. Nephrol Dial Transplant 1999;14:1229-33. Jansen JH, Høgasen K, Harboe M, Hovig T. In situ complement activation in porcine membranoproliferative glomerulonephritis type II. Kidney Int 1998;53:331-49. Cybulsky AV, Papillon J, McTavish AJ. Complement activates phospholipases and protein kinases in glomerular epithelial cells. Kidney Int 1998;54: 360-72. Nangaku M, Pippin J, Couser WG. Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome. J Am Soc Nephrol 1999;10:2323-31. Gallo-EG, Gianantonio-CA. Extrarenal involvement in diarrhoea-associated haemolytic uraemic syndrome. Pediatr Nephrol 1995;9:117-9. Sheth-KJ, Swick-HM, Haworth-N. Neurological involvement in hemolytic-uremic syndrome. Ann Neurol 1986; 19:90-3.
23. Petermann A, Offermann G, Distler A, Sharma AM. Familial hemolyticuremic syndrome in three generations. Am J Kidney Dis 1998;32:1063-7. 24. Mochon M, Kaiser BA, deChadarevian JP, Polinsky MS, Baluarte HJ. Cerebral infarct with recurrence of hemolytic-uremic syndrome in a child following renal transplantation. Pediatr Nephrol 1992;6:550-2. 25. Muller T, Sikora P, Offner G, Hoyer PF, Brodehl J. Recurrence of renal disease after kidney transplantation in children: 24 years of experience in a single center. Clin Nephrol 1998;49: 82-90. 26. Miller RB, Burke BA, Schmidt WJ, Gillingham KJ, Matas AJ, Mauer M, et al. Recurrence of haemolyticuraemic syndrome in renal transplants: a single-centre report. Nephrol Dial Transplant 1997;12:1425-30. 27. Karpman D, Lethagen S, Kristoffersson A, Isaksson C,.Holmberg L. von Willebrand factor mediates increased platelet retention in recurrent thrombotic thrombocytopenic purpura. Thromb Haemost 1997;78:1456-62. 28. Robson WLM, Leung AKC. The successful treatment of atypical hemolytic uremic syndrome with plasmapheresis. Clin Nephrol 1991;35:119-22. 29. Gianviti A, Perna A, Caringella A, Edefonti A, Penza R, Remuzzi G, et al. Plasma exchange in children with hemolytic uremic syndrome at risk of poor outcome. Am J Kidney Dis 1993; 22:264-6. 30. Fitzpatrick MM, Walters MDS, Trompeter RS, Dillon MJ, Barratt TM. Atypical (non diarrhea associated) hemolytic-uremic syndrome in childhood. J Pediatr 1993;122:532-7. 31. Høgasen K, Jansen JH, Molines TE, Hovdenes J, Harboe M. Hereditary porcine membranoproliferative glomerulonephritis type II is caused by factor H deficiency. J Clin Invest 1995;95:1054-61.
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Familial hemolytic uremic syndrome associated with complement factor H deficiency Daniel Landau, MD, Hannah Shalev, MD, Gal Levy-Finer, MD, Alexandra Polonsky, MD, Yael Segev, PhD, and Leonid Katchko, MD Atypical hemolytic uremic syndrome (HUS) associated with factor H deficiency (FHD) carries a poor prognosis. A 3-year-old girl with FHD-HUS reached end-stage renal disease at age 6 months after experiencing numerous relapses; she underwent a cadaveric renal transplant at age 46 months. One month after transplantation, she experienced an extensive non-hemorrhagic cerebral infarction. Later, hematologic and renal manifestations of HUS developed, followed by another massive cerebral infarction and death in spite of multiple plasma transfusions. A 14-month-old boy with FHDHUS experienced numerous HUS episodes starting at the age of 2 weeks. Daily plasma transfusions during relapses brought about only a temporary state of remission. However, prophylactic twice-weekly plasma therapy has been successful in preventing relapses and preserving renal function. With this regimen, serum factor H was increased from 6 mg/dL to subnormal values of 12 to 25 mg/dL (normal >60 mg/dL). We conclude that FHD-HUS recurs because FHD is not corrected by renal transplantation. A hypertransfusion protocol may prevent FHD-HUS. (J Pediatr 2001;138:412-7)
Atypical, diarrhea-negative forms of hemolytic uremic syndrome are a heterogeneous group of disorders. Inherited forms of HUS should be suspectFrom the Departments of Pediatrics and Pathology, Soroka University Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Submitted for publication May 26, 2000; revision received Sept 11, 2000; accepted Oct 26, 2000. Reprint requests: Daniel Landau, MD, Pediatric Nephrology, Department of Pediatrics “B,” Soroka Medical Center, PO Box 151, Beer Sheva 84101, Israel. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/112649 doi:10.1067/mpd.2001.112649
412
ed in any case of atypical HUS occurring among more than one family member on a non-epidemic basis.1 Some of these inherited forms of HUS have been associated with abnormalities in the complement system. Complement factor H, a 150-kd regulatory
See editorial, p 303. molecule of the complement cascade, plays a central role in the inhibition of the ongoing complement consumption in response to different stimuli.2 Recently, factor H deficiency has been implicated in the pathogenesis of atypical HUS.3,4 We have recently described an extended Bedouin family
with an autosomal recessive type of HUS, associated with severe FHD.5 This deficiency is due to a defective post-translational processing of a mutant factor H gene and is associated with complement consumption.6 Disease onset has been as early as 2 weeks of age, and the outcome has been very poor: all children have either died or reached end-stage renal disease by the end of the first year of life. No intervention, such as plasma infusion, plasma exchange, or intravenous immune globulin, has provided long-term efficacy in previous cases. In addition, no clear data exist regarding the risk for recurrence of this disease after renal transplantation. We report a patient with familial FHD-HUS who developed serious HUS after renal transplantation. In another patient, the efficacy of a novel therapy is presented. CNS ESRD FFP FHD HUS
Central nervous system End-stage renal disease Fresh frozen plasma Factor H deficiency Hemolytic uremic syndrome
METHODS Factor H measurement Serum samples were separated from whole blood and kept at –20°C until they were examined. Serum factor H levels were determined by using a radial immunodiffusion commercial kit (The Binding Site Co, Birmingham, UK), based on polyclonal antibodies in
LANDAU ET AL
THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 3 an agarose gel. The kit contains 3 calibrator solutions with predefined factor H concentrations (7, 42, and 70 mg/dL), to be used as internal controls. Five microliters of serum was applied to the agar plates and incubated for 72 hours in room temperature and controlled humidity conditions. The obtained rings’ radiuses were measured with a radial immunodiffusion plate reader (Grafar Co, Rockwell, Md) and compared with a reference table to determine factor H concentrations. All sample readings were done in duplicate.
Immunohistochemistry Immunohistochemical staining was carried out by the avidin-biotin-peroxidase complex method as previously described.7 An anti-human C5b-9 monoclonal antibody was used (Metra Biosystems, Mountain View, Calif). Deparaffinized rehydrated sections were incubated with nonimmune horse serum, followed by incubation with the monoclonal primary antibody (5 µg/mL) and biotin-labeled horse antimouse immunoglobulin. Then, an avidin-biotin-peroxidase complex was added, and this was visualized by addition of chromogen (DAB-H2O2 solution), producing a brown color of the reaction product. The sections were counterstained with hematoxylin. Negative control kidney biopsy slides (from a patient with mild proteinuria, with no histologic changes as determined by light microscopy and immunofluorescence) were treated similarly.
A
B
Case Descriptions
Fig 1. A, Electron microscopic (original magnification ×4500) photograph of allograft kidney biopsy specimen from patient 1, obtained on post-transplantation day 30, consistent with thrombotic microangiopathy. Glomerular capillary loop showing 2 swollen endothelial cells (asterisk) detached from the basement membrane.The subendothelial space shows fibrin deposition and platelet aggregation (open arrow).There is no evidence of immune complexes around the capillary basement membrane. Epithelial cell podocytes are effaced. B, Immunohistochemical staining of the allograft kidney biopsy specimen for C5b-9 (terminal attack complex). A part of the glomerulus is shown (original magnification ×800), with intracapillary fibrin thrombi (black arrows). C5b-9 immunostains positively in capillary walls (open arrows) and mesangium (asterisk).
CASE 1. A 46-month-old Bedouin girl was found at birth to have low serum C3 levels (250 mg/L). She was the fourth child of consanguineous parents, members of an extended Bedouin family with hypocomplementemic HUS associated with FHD, previously described.5 Her two younger sisters died of familial HUS at the ages of 2 weeks and 3 months. Complete blood count and serum urea and creatinine levels were normal after birth. At age 3
weeks, she was admitted to the hospital for the first time because of vomiting and lethargy. Microangiopathic hemolytic anemia (plasma hemoglobin of 8.4 g/100 mL, schistocytes, reticulocytosis, thrombocytopenia (102,000) and renal insufficiency (increases in creatinine and urea nitrogen values to 114.9 µmol/L [1.3 mg/100 mL] and 8.56 mmol/L [24 mg/100 mL], respectively)
were observed. Hypocomplementemia (C3: 370 mg/L) persisted. Serum factor H levels were very low (<6 mg/100 mL; normal >60).8 Findings on a renal biopsy specimen obtained at age 3 weeks were consistent with HUS, as previously described.5 She was treated with daily doses of fresh frozen plasma (10 mL/kg) for 3 days, which caused a transient clinical improvement, be413
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Fig 2. Brain computed tomography of patient 1 shows extensive parietal infarctions on the distribution of both middle cerebral arteries. In addition, an area of right subacute ischemia with “luxury perfusion” (arrow), as well as a left subacute ischemia with basal ganglia involvement, is seen. An old left posterior cerebral infarction on the distribution of the posterior cerebral artery (arrowhead) is also seen.
cause shortly thereafter she developed recurrent HUS relapses (none of which were associated with central nervous system involvement). No other specific therapy was provided. At age 6 months, peritoneal dialysis was initiated for anuric ESRD. She did not experience further HUS relapses as ESRD developed. Serum C3 levels during ESRD remained low. At age 44 months, she underwent cadaveric renal transplantation. The postoperative course was uneventful: urine output was adequate, and serum creatinine concentration decreased from 419.9 µmol/L (4.8 mg/100 mL) to 19.4 µmol/L (.2 mg/100 mL) within days. The patient received tacrolimus, azathioprine, and prednisone. In addition, she received low-dose aspirin, ranitidine, and prophylactic co-trimoxazole. Her blood pressure was well controlled with atenolol, minoxidil, and clonidine. On post-transplantation day 16, a left hemiparesis was noted. Her blood 414
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Fig 3. Patient 2: serum creatinine (1000*mg/dL) (open squares) and LDH (U/L) (black circles) levels since birth. Blood transfusions (arrows) and elevation of serum creatinine and LDH levels reflect a state of HUS relapse. LDH, Lactate dehydrogenase; FFP PRN, fresh frozen plasma (10 mL/kg/dose) given daily during relapses; IFN-γ, interferon-γ (2.5 U/kg/dose, 3 times a week); FFP qw, FFP (15 mL/kg/dose) given as prophylaxis once a week; FFP BIW, FFP (15-20 mL/kg/dose) given as prophylaxis twice a week. pressure was well controlled (110/70 mm Hg). A large ischemic cerebral infarct extending along the right middle cerebral artery was demonstrated on brain computerized tomography. A workup for stroke predisposing conditions included search for bleeding tendency and/or thrombophilic states, none of which were present. Echocardiography demonstrated left ventricular hypertrophy and moderate mitral regurgitation without evidence of atrial thrombi. Two weeks later (1 month after transplantation), a deterioration in renal function was noted. Serum creatinine and urea concentrations increased from 17.6 µmol/L (.2 mg/100 mL) and 5.7 mmol/L (16 mg/100 mL) to 83 µmol/L (.94 mg/100 mL) and 27.1 mmol/L (76 mg/100 mL), respectively. A microangiopathic anemia picture was evident, and hypertension developed. A percutaneous kidney biopsy specimen revealed a histologic picture consistent with HUS relapse (Fig 1, A): fibrin thrombi filled the glomerular capillary lumina, and there was a segmental collapse of glomerular membranes. Electron microscopy
showed focal necrosis of endothelial cells and fibrin thrombi in glomerular capillary lumina but no evidence of immune complex deposition. No glomeruli were available for immunofluorescent investigation. Immunohistochemistry with a C5b-9 monoclonal antibody showed specific staining for this protein in glomerular capillary loops and mesangium (Fig 1, B). This staining was absent in a kidney biopsy specimen from a patient with minimal change disease. Mild evidence of tubulointerstitial rejection was also seen, with infiltration of mononuclear cells inside the venular walls. The child became stuporous, tachypneic, and pale. Repeat brain computed tomography (Fig 2) showed a new finding in the left cerebral hemisphere with the same diffuse low-density appearance previously noted on the right, consistent with a second ischemic cerebral infarct. A therapeutic trial with FFP did not prove effective. Neurologic examination showed evidence of brain death, and she died 3 days later, 34 days after transplantation. The family did not permit a postmortem examination.
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THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 3 CASE 2. A term male infant was found at birth to have low C3 levels (220 mg/L). Four previous siblings died of HUS and were previously reported.5 His serum creatinine and hemoglobin values were normal at birth, but at age 10 days, he had a first episode of HUS, manifested as renal failure (creatinine, 274 µmol/L, 3.1 mg/100 mL), hypertension, and elevated lactate dehydrogenase levels. He was treated with daily transfusions of FFP, 10 mL/kg, until signs of remission developed (Fig 3). However, he later experienced numerous relapses of the disease, with abnormal levels of serum creatinine, uric acid, lactate dehydrogenase, and triglycerides (increased during relapse), and hemoglobin (decreased during relapse). Blood pressure remained elevated, and left ventricular hypertrophy was evident at age 7 months. He required 16 blood transfusions over the first 8 months of life for repeated episodes of anemia (hemoglobin <1 g/100 mL). Serum factor H levels were very low (<6 mg/100 mL, normal >60). A trial with subcutaneous interferon-γ (2.5 U/kg/dose, three times a week), reported to increase factor H synthesis by hepatocytes,9 was given for 2 months. Factor H levels were only mildly elevated from baseline (8.2 mg/100 mL), but no long-term remission was achieved. A subsequent trial of weekly prophylactic FFP transfusion (10 mL/kg/dose) was also unsuccessful in preventing further relapses. At age 8.5 months, a twice-a-week regimen of FFP, 15 to 20 mL/kg/dose (from a single-donor pheresis source), administered through an implanted central venous catheter, was initiated. This treatment brought about a persistent decrease in serum creatinine concentration (26.5-39.8 µmol/L, 0.3-0.45 mg/100 mL) and a lower demand for blood transfusions (only one in the 7 months since the beginning of this intervention) (Fig 3). Blood pressure was better controlled, and left ventricular hypertrophy gradually regressed.
Fig 4. Serum factor H levels of patient 2 before any regular intervention (“zero”) and timely during a twice-weekly (Mondays and Thursdays) 20 mL/kg FFP protocol: 0.5, 24, 48, and 72 hours after finishing the transfusion.Three columns at right show factor H levels from patient’s (heterozygote) mother, healthy sibling, and infused FFP.
Factor H peak level after FFP administration was as high as 23 mg/100 mL. At trough, 72 hours after FFP administration, it was above 10 mg/100 mL. Factor H level in the transfused plasma sample was 61 mg/100 mL (Fig 4). Serum C3 levels remained low and did not change. At present, 9 months after the initiation of this treatment, there is no apparent increase in plasma requirements. Urinalysis shows no protein or blood.
DISCUSSION These cases add to our knowledge about the course of this unique form of atypical HUS. No further HUS relapses were encountered as soon as patient 1 progressed to ESRD. A similar long-term HUS remission was previously observed.5 Nevertheless, persistent hypocomplementemia indicated that the basic complement consumption process was ongoing.6 Even
though FHD may cause ongoing complement consumption, the causative role of complement consumption in HUS is not understood. The endothelium is the principal target tissue in HUS, and endothelial cells express factor H.10 The permissive role of the kidney endothelial bed in the pathogenesis of HUS has been previously shown in a group of adult patients with refractory HUS in whom remission was achieved only after bilateral nephrectomy.11 The fact that this atypical HUS variant “needs” functioning kidney tissue for its propagation is well portrayed in case 1: HUS recurred as soon as new functioning kidney tissue was introduced. Plasma obtained from patients with thrombotic thrombocytopenic purpura and patients with atypical HUS induce apoptosis and expression of the apoptosis-associated molecule Fas (CD95) only in small vessel dermal, renal, and cerebral lineages but not in pulmonary or hepatic lineages of microvascular endothelial 415
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cells.12 This dichotomy parallels the current clinical observation and supports the existence of organ-specific endothelial cells with different susceptibilities to injury. However, further evidence is needed to prove this hypothesis. FHD13 has been associated with several diseases, such as systemic lupus erythematosus,14 type II membranoproliferative glomerulonephritis,15 a form of chronic collagen type III glomerulopathy,16 and atypical familial HUS of both recessive5 and dominant17 inheritance. We have previously shown that this specific FHD-HUS is associated with very low serum factor H levels and is caused by a mutation in the factor H gene on chromosome 1. The defective protein product of the mutated factor H gene can be detected intracellularly but is not secreted by cultured fibroblasts.6 Factor H– deficient Norwegian pigs develop ESRD with pathologic characteristics of membranoproliferative glomerulonephritis.18 These animals have congenital FHD, associated with hypocomplementemia early in life and subsequent death from renal failure caused by massive complement deposition in glomeruli. Although no immune complex disease developed in our patients, C5b-9 was present in capillary loops in the child with post-transplantation HUS. No such immunostaining could be detected in normal kidney tissue. Complement activation on platelet-endothelial interaction is mediated by C5b-9 production on the endothelial surface, associated with a shift toward a more procoagulant state. In addition, it is now known that the response to C5b-9 injury is not merely due to the creation of holes in the cell membrane but may also be due to a “sublytic” effect.19 This phenomenon may have important influence in different renal diseases.20 However, further studies are needed to determine whether this C5b-9 staining in the damaged capillaries in our case is a specific phenomenon for FHD-HUS 416
THE JOURNAL OF PEDIATRICS MARCH 2001 or merely reflects a complement-mediated injury to endothelium. Overall, the absence of this circulating factor leads to kidney endothelial damage and ESRD. Renal transplantation does not correct this extrarenal deficiency, and therefore HUS relapses after this intervention. CNS involvement in HUS has been described for both the typical21,22 and atypical forms23 in up to 30% of cases. CNS involvement as a preliminary sign of post-transplantation HUS relapse has been previously reported.24 This was also a young patient with atypical HUS who did not exhibit any CNS involvement during the HUS episode that led to ESRD. HUS relapse in patients who have undergone renal transplantation has been previously described (mainly the atypical forms).25,26 CNS involvement has not occurred in the previous 11 children with primary FHD-HUS who have been treated (but have not undergone transplantation) and died in our institution in the past 20 years.5 Perhaps the severe CNS involvement was related to extensive endothelial damage, occurring when a large adult kidney was transplanted into a small child. This may have allowed the accumulation of abnormal von Willebrand factor multimers, which facilitates the deposition of platelet microthrombi.27 No therapy proved effective in the 11 patients that we treated in the past 20 years, including transfusions of FFP during relapses, intravenous immune globulin, or plasmapheresis. This is at variance with other reports28,29 that claim a therapeutic benefit of plasma therapy in atypical HUS. Plasmapheresis was effective in inducing remission and extending renal survival.30 However, the number of relapses was even higher, again suggesting that a circulating factor is only temporarily replaced (or removed) by plasma therapy (or exchange). Case 2 shows that FHD-HUS can be prevented by frequent (twice weekly) plasma therapy, when a partial correction of serum fac-
tor H levels occurs. The twice-a-week FFP transfusion protocol was adopted based on the experience with factor H–deficient Norwegian pigs. When these piglets were given weekly plasma transfusions, their survival was extended.31 In our patient, the plasma hypertransfusion protocol was able to increase serum factor H levels to only subnormal values, yet it was sufficient to prevent clinical relapses. C3 levels remain depressed with this protocol, suggesting that complement consumption continues. In addition, renal biopsy specimens from patients with FHDHUS do not show evidence of massive complement deposition,5 contrary to the porcine FHD model. Thus FFP transfusions, which increase serum factor H levels, are beneficial in preventing HUS relapses in spite of persistent low C3 levels, suggesting again a possible independent, protective role for factor H, irrespective of ongoing complement activation. Based on this new observation, in future cases of patients with FHD reaching ESRD that requires renal transplantation, preemptive and posttransplantation factor H replacement should be considered. This can currently be done by repeated plasma transfusions, to be started together with the grafting surgical procedure.
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