- Email: [email protected]
Distal Angiopathy and Atypical Hemolytic Uremic Syndrome: Clinical and Functional Properties of an Anti–Factor H IgAλ Antibody
Case Report Distal Angiopathy and Atypical Hemolytic Uremic Syndrome: Clinical and Functional Properties of an Anti–Factor H IgAl Antibody Claire Rigothier, MD, PhD,1,2 Yahsou Delmas, MD, PhD,1 Lubka T. Roumenina, PhD,3,4,5 Ce´cile Contin-Bordes, PhD,6 Se´bastien Lepreux, MD, PhD,2,7 Frank Bridoux, MD, PhD,8,9 Jean Michel Goujon, MD, PhD,9,10 Thomas Bachelet, MD, PhD,1 Guy Touchard, MD, PhD,8,9 Ve´ronique Fre´meaux-Bacchi, MD, PhD,3,11 and Christian Combe, MD, PhD1,2 Abnormal regulation of the alternative pathway of the complement system is a well-described trigger of microangiopathy leading to atypical hemolytic uremic syndrome (aHUS). However, the involvement of complement dysregulation in distal angiopathy has not been reported in adults. We describe the clinical course of a patient with severe distal angiopathy (amputation of all fingers and toes) followed 3 years later by aHUS with end-stage renal disease. This course was attributed to a circulating monoclonal immunoglobulin A l light chain (IgAl) with unusual properties: it bound complement factor H (CFH) and impaired CFH-glycosaminoglycan interaction and cell-surface protection. Local complement activation with distal angiopathy and microvascular injury was suggested by deposition of IgA, C4d, and C5b-9 in limb and preglomerular arteries. We therefore postulated that the monoclonal IgAl inhibited activity of endothelial cell–bound CFH, which led to local activation of complement, vasoconstriction (distal angiopathy), and aHUS. While the patient was dependent on dialysis and plasma exchange, treatment with the anti-C5 antibody eculizumab induced remission of distal angiopathy and aHUS. During eculizumab treatment, kidney transplantation was performed. The patient had normal kidney function at the 3-year follow-up. We suggest that the association of distal angiopathy and aHUS in this patient is clearly linked to anti-CFH properties of the monoclonal IgAl. Am J Kidney Dis. -(-):---. ª 2015 by the National Kidney Foundation, Inc. INDEX WORDS: Gammopathy; autoantibody; alternative complement pathway; complement activation; complement factor H (CFH); factor H antibody; macroangiopathy; distal angiopathy; atypical hemolytic uremic syndrome (aHUS); microvascular injury.
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veractivation of the alternative pathway of the complement system may lead to atypical hemolytic uremic syndrome (aHUS).1-3 Complement activation has also been described in diseases involving middle and large vessels such as myocardial infarction and age-related macular degeneration.4 To our knowledge, no link has been established to date in adults between distal angiopathic insults and a complement-induced microvascular injury such as aHUS. In this report, we describe the clinical course of a patient in whom a monoclonal anti–complement factor H (CFH) immunoglobulin A (IgA) l autoantibody led
to severe distal angiopathy. This was followed 3 years later by aHUS, a form of microvascular injury.
From the 1Service de Néphrologie Transplantation Dialyse, Centre Hospitalier Universitaire de Bordeaux; 2INSERM U1026, Université de Bordeaux, Bordeaux; 3Complement and Diseases Team, INSERM UMRS 1138, Cordeliers Research Center; 4Université Paris Descartes Sorbonne Paris-Cité; 5Université Pierre et Marie Curie (UPMC-Paris-6), Paris; 6Laboratoire d’Immunologie and 7Laboratoire de Pathologie, Centre Hospitalier Universitaire de Bordeaux, Bordeaux; 8Service de Néphrologie, Hémodialyse et Transplantation Rénale, Centre Hospitalier Universitaire de Poitiers; 9Centre de référence des amyloses primitives et des maladies de dépôts d’immunoglobulines monoclonales; 10Laboratoire d’Anatomie et cytologie Pathologiques, Centre Hospitalier Universitaire de
Poitiers, Poitiers; and 11Assistance Publique-Hopitaux de Paris, Service d’Immunologie biologique, Hôpital Européen Georges Pompidou, Paris, France. Received November 27, 2014. Accepted in revised form March 11, 2015. Address correspondence to Claire Rigothier, MD, PhD, Hôpital Pellegrin, Service de Néphrologie Transplantation Dialyse, Place Amélie Raba Léon, 33076 Bordeaux, France. E-mail: claire. [email protected] 2015 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2015.03.039
Am J Kidney Dis. 2015;-(-):---
CASE REPORT A 45-year-old white man developed severe Raynaud-like phenomenon. The disease presented as flares with livedo, mottling, cyanosis, and severe morphine-resistant pain of the extremities. After several months, trophic lesions developed leading to the amputation of one finger 2 years later. No infectious, autoimmune, endocrine, hematologic, or vascular disease was identified. Distal necrosis worsened, and all fingers and toes were amputated. Several months after these amputations, the patient experienced a severe flare, with reopening of wounds and vascular purpura on his legs (Fig S1, available as online supplementary material).
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Figure 1. Pathology studies. (A) Preglomerular vessels presented intracapillary obstruction (/) associated with fibroproliferative endarteritis. In glomeruli, thickening or splitting of capillary walls (➤) was observed. These abnormalities were associated with swelling of subendothelial space and mesangial widening (*). (B) Electron microscopy was typical of atypical hemolytic uremic syndrome lesions in glomeruli. Immunofluorescence imaging of immunoglobulin A deposits in the (C) preglomerular vessels and (D) limb small arteries, C4d deposits in the (E) preglomerular vessels and (F) limb small arteries, and deposits of (G) C5b9 and (H) factor H in the renal microvasculature.
Hemolytic anemia, thrombopenia, and rapidly progressive kidney failure led to the diagnosis of HUS. Infections, autoimmune diseases, and malignancies are all potential causes of HUS; however, each of these possible causes was ruled out in this case. 2
A kidney biopsy specimen showed multiple intracapillary glomerular thrombi and vascular lesions characteristic of HUS (Fig 1A and B), as well as IgA, C4d, C5b-9, and CFH deposits on preglomerular vessels (Fig 1C, E, G, and H). No Congo Red staining was detectable Am J Kidney Dis. 2015;-(-):---
Anti-FH IgA With aHUS and Distal Angiopathy
Figure 2. Effects of the immunoglobulin A (IgA) anti–complement factor H (CFH) antibody on the cell-surface protection capacity of CFH. (A) Nonsensitized sheep erythrocytes were incubated with increasing concentrations of plasma from the patient (samples from 2 different dates) and 2 healthy controls (C1, C2) or pooled control plasma in the presence of a buffer (containing magnesium EGTA) that favors the alternative complement pathway. Spontaneous lysis was assessed by released hemoglobin, as previously described.9 (B) Flow cytometric analysis of C3 deposition on activated and late apoptotic human umbilical vein endothelial cells (HUVECs). Primary HUVECs (passage 3) were cultured and treated as previously described.1,2 Briefly, resting, activated (stimulated with TNF-a and IFNg), and late apoptotic cells were incubated with 33% patient serum or healthy human serum, diluted in M199 medium. After detachment, cells were labeled to assess C3 deposition using a monoclonal anti-C3c antibody (Quidel) and staining was detected by flow cytometry. Black and solid gray lines denote HUVECs incubated with patient and control serum, respectively. Irrelevant IgG1 was used as an isotype control (dashed gray line). (C) Apoptotic HUVECs were analyzed as in panel B, except that serum was supplemented with 0, 50, 150, or 300 mg/mL of purified CFH (Comptech) prior to being added to the cells. (D) Fast protein liquid chromatography (FPLC) eluate fractions from a heparin affinity column loaded with control or patient plasma were probed by Western blotting for the presence of CFH, using polyclonal anti-CFH antiserum (which also recognizes CFH-related proteins 1, 2, and 5. The smaller band was presumably CFHR5. (E, F) Dilutions of control and patient plasma, adjusted to ensure the same amount of CFH, were incubated
Am J Kidney Dis. 2015;-(-):---
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Rigothier et al and mesangial IgA deposits were not observed. Reassessment of specimens from the previously amputated fingers and toes also found IgA and C4d deposits on small-artery walls (Fig 1D and F). Immunofixation of plasma and urine samples revealed a monoclonal IgAl. The serum IgAl monoclonal gammopathy was also detected upon retrospective immunofixation analysis of a sample taken shortly before the finger/toe amputations. Bone marrow smears and a biopsy specimen showed that 4% of cells were plasma cells, which stained positive with anti-l and anti-a antibodies. CH50, C3, C4, factor B, CFH, factor I, and CD46 (membrane cofactor protein) levels were unremarkable (Fig S2A). Genetic analyses detected no mutations in the genes coding for CFH, factor I, CD46, factor B, and C3; however, the patient was homozygosis for a complete deletion of the CFHR1/CFHR3 locus.5 Examination of the patient’s plasma detected binding of the IgA autoantibody to CFH (Fig S2B). His plasma strongly induced lysis of nonsensitized sheep erythrocytes (Fig 2A) and enhanced C3 deposition on activated or late apoptotic, but not on resting, endothelial cells (Fig 2B). In side-by-side tests of the patient’s serum and serum from a healthy control, addition of purified CFH resulted in a decrease in C3 deposition on late apoptotic cells; however, twice as much CFH had to be added to the patient’s serum to reach 50% inhibition (Fig 2C). The interaction of CFH with the model glycosaminoglycan heparin was measured by affinity chromatography of plasma from the patient or a healthy control. CFH eluted from the control plasma appeared at fractions 25 to 29, while no CFH was present in the corresponding fractions from the patient’s plasma, suggesting that CFH from the patient’s plasma does not interact with heparin (Fig 2D). The patient’s anti-CFH autoantibody did not affect the binding of plasma CFH to C3b (Fig 2E) or C3d (Fig 2F) and did not perturb the dissociation of C3 convertase, as measured by a specific hemolytic assay (Fig 2G). Plasma exchange therapy was started, and then after 1 month, corticosteroid treatment was begun. Despite an initial improvement in clinical and biological parameters, the patient experienced multiple aHUS relapses. Throughout the patient’s course, there was a clear-cut temporal association between symptoms of distal angiopathy and biological signs of aHUS. Several therapeutic strategies were used without obvious efficacy against aHUS symptoms. Concomitant with aHUS relapses, kidney function deteriorated and hemodialysis therapy was started 5 years after initial presentation. The time course of treatments and the evolution of aHUS parameters, serum IgA levels, and kidney function are summarized in Fig S3. Five months after dialysis therapy initiation, eculizumab therapy was started, in the setting of a clinical trial of eculizumab in aHUS (ClinicalTrials.gov identifier: NCT00838513).6 The effect of eculizumab was impressive, with immediate and definitive remission of clinical symptoms and biological signs of distal angiopathy and thrombotic microangiopathy. The patient reported a drastic improvement in quality of life. He remained hemodialysis dependent for a further 7 months, then received a deceased donor kidney transplant. He continued eculizumab treatment (with supplemental administration after surgery) and experienced an uneventful time course, with no relapse of distal angiopathy or aHUS.
DISCUSSION To our knowledge, this is the first case report of anti-CFH IgA monoclonal gammopathy–associated
aHUS with severe distal angiopathy. We present evidence that the anti-CFH IgAl that was detected was functionally relevant because it inhibited CFH binding to glycosaminoglycans in vitro and impaired cell-surface protection in cell-based assays. We have previously shown that anti-CFH antibodies of the IgG isotype (directed against multiple CFH epitopes located at the amino and carboxy termini) perturbed CFH-mediated cell protection, inhibited CFH interaction with C3, and caused C3 consumption.7 In the present case, the anti-CFH activity of the patient’s IgAl antibody blocked the interaction of CFH with the cell surface. In vitro, the patient’s plasma strongly induced lysis of nonsensitized sheep erythrocytes, as is also observed in plasma from patients with CFH mutations or anti-CFH IgG autoantibodies. This effect is not observed in plasma from healthy donors.8,9 The antibodies did not perturb the functional activity of CFH toward associated C3 convertase because C3 levels remained normal throughout the disease course. Moreover, an increase of C3 deposition on cultured endothelial cells was detected from the patient’s serum as compared to control serum. This result is similar to a previous report for CFHdepleted serum or to cases of C3 gene mutations.1 Pathologic evidence of local complement activation and IgA deposition was found in the small arteries of the kidney and also those of the amputated fingers and toes. We hypothesize that binding of IgAl to CFH inhibits the interaction of CFH with glycosaminoglycans on endothelial cells, leading to local complement activation. Release of the anaphylatoxins C3a and C5a could have induced vasoconstriction, inflammation, and thrombosis of large vessels, resulting in the various clinical manifestations and finally distal necrosis. This clinical case is exceptional due to the time course of the disease (late in adult life with distal angiopathy 3 years before aHUS), the unique pathophysiology linked to the properties of the monoclonal IgAl, and the favorable outcome after eculizumab therapy. Distal angiopathy or extrarenal manifestations have been rarely reported in aHUS, and always in children.10 There are published reports of 4 children who presented with gangrenous lesions of the distal phalanges of several fingers and toes after initial presentation with aHUS.11-13 In addition, 4 children who presented with large-artery stenosis and end-stage renal disease have been reported in the literature.14,15
with immobilized (E) C3b or (F) C3d. The amount of bound CFH was detected by ELISA. (G) Increasing concentrations of the patient’s plasma (samples from 2 different dates), pooled control plasma, or plasma from an individual with CFH haploinsufficiency (FH HI) were added to sheep erythrocytes with preformed C3 convertases to dissociate the C3bBb complexes. The residual convertases present on the cell surface were measured after addition of terminal complement components by degree of cell lysis (detected spectrophotometrically at 414 nm by the released hemoglobin). 4
Am J Kidney Dis. 2015;-(-):---
Anti-FH IgA With aHUS and Distal Angiopathy
Loirat et al16 described the development of stenoses of several thoracic and cerebral arteries in a child on longterm dialysis therapy with a factor B mutation, several years after the first flare of aHUS. In a case reported in 2009, hyperactivation of the C3 convertase with C3 fragment deposition at the endothelial surface was probably responsible for a proinflammatory and thrombogenic phenotype leading to small- and largeartery stenoses.2 All these cases of distal angiopathy involved children with very severe forms of aHUS and early onset of the disease; to our knowledge, no case has been reported in adults. With regard to extrarenal complications, these have been described in .20% of patients with aHUS.17 In the large series of patients with aHUS18 from an Indian registry, only 2 of 101 developed peripheral gangrene soon after presentation.11,19 Anti-CFH antibody–associated aHUS primarily affects children between 9 and 13 years old, but can also affect adults. The anti-CFH isotype is typically IgG, but Strobel et al20 reported 3 patients with antiCFH IgA autoantibodies. Dragon-Durey et al17 have reported 7 cases of anti-CFH antibody associated with aHUS of adult onset, but none of the patients presented with monoclonal gammopathy. In this patient, IgA monoclonal gammopathy was detected retrospectively in the patient’s serum early in the history of distal angiopathy and before aHUS onset. We postulate that monoclonal IgAl is the pathophysiologic link between distal angiopathy and aHUS through chronic local complement dysregulation.21 Due to the patient’s presentation with monoclonal gammopathy of undetermined significance with a low level of circulating IgA, we opted to treat him with eculizimab (as we would any patient with aHUS).22 The dramatic effects of eculizumab on ischemic symptoms is another strong argument for complement overactivation in the pathophysiology of distal angiopathy.23 We hypothesize that the inhibition of C5 activity inhibited the release of C5a and activation of the terminal complement pathway, thereby protecting endothelial cells and limb arteries, with disappearance of distal angiopathy symptoms. In conclusion, we present a novel observation of monoclonal IgAl with anti-CFH activity leading to severe distal angiopathy and aHUS.
ACKNOWLEDGEMENTS We thank the patient for providing informed consent to permit publication of these data. Support: This study was supported by grants from the Agence Nationale de la Recherche (ANR Genopath 2009-2012 09geno03101I) and by EU FP7 grant 2012-305608 (EURenOmics) to Dr Frémeaux-Bacchi. Financial Disclosure: Drs Delmas and Combe have received speaker fees from Alexion. The authors declare that they have no other relevant financial interests. Am J Kidney Dis. 2015;-(-):---
SUPPLEMENTARY MATERIAL Figure S1: Photographs of the lower extremities during flare. Figure S2: Analysis of complement pathway components and detection of anti-CFH IgA. Figure S3: Time course of treatments and eGFR, LDH, and serum IgA levels. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2015.03.039) is available at www.ajkd.org
REFERENCES 1. Roumenina LT, Frimat M, Miller EC, et al. A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function. Blood. 2012;119(18):4182-4191. 2. Roumenina LT, Jablonski M, Hue C, et al. Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome. Blood. 2009;114(13):2837-2845. 3. Manuelian T, Hellwage J, Meri S, et al. Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome. J Clin Invest. 2003;111(8):1181-1190. 4. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308(5720):385-389. 5. Jozsi M, Licht C, Strobel S, et al. Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/ CFHR3 deficiency. Blood. 2008;111(3):1512-1514. 6. Legendre CM, Licht C, Muus P, et al. Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368(23):2169-2181. 7. Blanc C, Roumenina LT, Ashraf Y, et al. Overall neutralization of complement factor H by autoantibodies in the acute phase of the autoimmune form of atypical hemolytic uremic syndrome. J Immunol. 2012;189(7):3528-3537. 8. Sanchez-Corral P, Perez-Caballero D, Huarte O, et al. Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome. Am J Hum Genet. 2002;71(6):1285-1295. 9. Roumenina LT, Roquigny R, Blanc C, et al. Functional evaluation of factor H genetic and acquired abnormalities: application for atypical hemolytic uremic syndrome (aHUS). Methods Mol Biol. 2014;1100:237-247. 10. Fremeaux-Bacchi V, Fakhouri F, Garnier A, et al. Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults. Clin J Am Soc Nephrol. 2013;8(4):554-562. 11. Malina M, Gulati A, Bagga A, Majid MA, Simkova E, Schaefer F. Peripheral gangrene in children with atypical hemolytic uremic syndrome. Pediatrics. 2013;131(1):e331-e335. 12. Ozel A, Caliskan U, Gucer S. Peripheral gangrene complicating hemolytic uremic syndrome in a child. Pediatr Nephrol. 2003;18(5):465-467. 13. Kaplan BS, Garcia CD, Chesney RW, Segar WE, Giugno K, Chem R. Peripheral gangrene complicating idiopathic and recessive hemolytic uremic syndromes. Pediatr Nephrol. 2000;14(10-11):985-989. 14. Davin JC, Gracchi V, Bouts A, Groothoff J, Strain L, Goodship T. Maintenance of kidney function following treatment with eculizumab and discontinuation of plasma exchange after a third kidney transplant for atypical hemolytic uremic syndrome associated with a CFH mutation. Am J Kidney Dis. 2010;55(4): 708-711. 5
Rigothier et al 15. Vergouwen MD, Adriani KS, Roos YB, Groothoff JW, Majoie CB. Proximal cerebral artery stenosis in a patient with hemolytic uremic syndrome. AJNR Am J Neuroradiol. 2008;29(5): e34. 16. Loirat C, Macher MA, Elmaleh-Berges M. Non-atheromatous arterial stenoses in atypical haemolytic uraemic syndrome associated with complement dysregulation. Nephrol Dial Transplant. 2010;25:3421-3425. 17. Dragon-Durey MA, Sethi SK, Bagga A, et al. Clinical features of anti-factor H autoantibody-associated hemolytic uremic syndrome. J Am Soc Nephrol. 2010;21(12):2180-2187. 18. Herrmann SM, Govani MV, Fidler ME, et al. Recurrence of monoclonal IgA lambda glomerulonephritis in kidney allograft associated with multiple myeloma [published online ahead of print November 5, 2014]. Clin Nephrol. http://dx.doi.org/10.5414/CN1 08339. 19. Sinha A, Gulati A, Saini S, et al. Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of antifactor H autoantibody-associated hemolytic uremic syndrome
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in children [published online ahead of print October 2, 2013]. Kidney Int. http://dx.doi.org/10.1038/ki.2013.373. 20. Strobel S, Abarrategui-Garrido C, Fariza-Requejo E, Seeberger H, Sanchez-Corral P, Jozsi M. Factor H-related protein 1 neutralizes anti-factor H autoantibodies in autoimmune hemolytic uremic syndrome. Kidney Int. 2011;80(4):397-404. 21. Jokiranta TS, Solomon A, Pangburn MK, Zipfel PF, Meri S. Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H. J Immunol. 1999;163(8):4590-4596. 22. Cheungpasitporn W, Leung N, Sethi S, Gertz MA, Fervenza FC. Refractory atypical hemolytic uremic syndrome with monoclonal gammopathy responsive to bortezomib-based therapy [published online ahead of print October 27, 2014]. Clin Nephrol. http://dx.doi.org/10.5414/CN108363. 23. Zuber J, Fakhouri F, Roumenina LT, Loirat C, FremeauxBacchi V. Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat Rev Nephrol. 2012;8(11): 643-657.
Am J Kidney Dis. 2015;-(-):---
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veractivation of the alternative pathway of the complement system may lead to atypical hemolytic uremic syndrome (aHUS).1-3 Complement activation has also been described in diseases involving middle and large vessels such as myocardial infarction and age-related macular degeneration.4 To our knowledge, no link has been established to date in adults between distal angiopathic insults and a complement-induced microvascular injury such as aHUS. In this report, we describe the clinical course of a patient in whom a monoclonal anti–complement factor H (CFH) immunoglobulin A (IgA) l autoantibody led
to severe distal angiopathy. This was followed 3 years later by aHUS, a form of microvascular injury.
From the 1Service de Néphrologie Transplantation Dialyse, Centre Hospitalier Universitaire de Bordeaux; 2INSERM U1026, Université de Bordeaux, Bordeaux; 3Complement and Diseases Team, INSERM UMRS 1138, Cordeliers Research Center; 4Université Paris Descartes Sorbonne Paris-Cité; 5Université Pierre et Marie Curie (UPMC-Paris-6), Paris; 6Laboratoire d’Immunologie and 7Laboratoire de Pathologie, Centre Hospitalier Universitaire de Bordeaux, Bordeaux; 8Service de Néphrologie, Hémodialyse et Transplantation Rénale, Centre Hospitalier Universitaire de Poitiers; 9Centre de référence des amyloses primitives et des maladies de dépôts d’immunoglobulines monoclonales; 10Laboratoire d’Anatomie et cytologie Pathologiques, Centre Hospitalier Universitaire de
Poitiers, Poitiers; and 11Assistance Publique-Hopitaux de Paris, Service d’Immunologie biologique, Hôpital Européen Georges Pompidou, Paris, France. Received November 27, 2014. Accepted in revised form March 11, 2015. Address correspondence to Claire Rigothier, MD, PhD, Hôpital Pellegrin, Service de Néphrologie Transplantation Dialyse, Place Amélie Raba Léon, 33076 Bordeaux, France. E-mail: claire. [email protected] 2015 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2015.03.039
Am J Kidney Dis. 2015;-(-):---
CASE REPORT A 45-year-old white man developed severe Raynaud-like phenomenon. The disease presented as flares with livedo, mottling, cyanosis, and severe morphine-resistant pain of the extremities. After several months, trophic lesions developed leading to the amputation of one finger 2 years later. No infectious, autoimmune, endocrine, hematologic, or vascular disease was identified. Distal necrosis worsened, and all fingers and toes were amputated. Several months after these amputations, the patient experienced a severe flare, with reopening of wounds and vascular purpura on his legs (Fig S1, available as online supplementary material).
1
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Figure 1. Pathology studies. (A) Preglomerular vessels presented intracapillary obstruction (/) associated with fibroproliferative endarteritis. In glomeruli, thickening or splitting of capillary walls (➤) was observed. These abnormalities were associated with swelling of subendothelial space and mesangial widening (*). (B) Electron microscopy was typical of atypical hemolytic uremic syndrome lesions in glomeruli. Immunofluorescence imaging of immunoglobulin A deposits in the (C) preglomerular vessels and (D) limb small arteries, C4d deposits in the (E) preglomerular vessels and (F) limb small arteries, and deposits of (G) C5b9 and (H) factor H in the renal microvasculature.
Hemolytic anemia, thrombopenia, and rapidly progressive kidney failure led to the diagnosis of HUS. Infections, autoimmune diseases, and malignancies are all potential causes of HUS; however, each of these possible causes was ruled out in this case. 2
A kidney biopsy specimen showed multiple intracapillary glomerular thrombi and vascular lesions characteristic of HUS (Fig 1A and B), as well as IgA, C4d, C5b-9, and CFH deposits on preglomerular vessels (Fig 1C, E, G, and H). No Congo Red staining was detectable Am J Kidney Dis. 2015;-(-):---
Anti-FH IgA With aHUS and Distal Angiopathy
Figure 2. Effects of the immunoglobulin A (IgA) anti–complement factor H (CFH) antibody on the cell-surface protection capacity of CFH. (A) Nonsensitized sheep erythrocytes were incubated with increasing concentrations of plasma from the patient (samples from 2 different dates) and 2 healthy controls (C1, C2) or pooled control plasma in the presence of a buffer (containing magnesium EGTA) that favors the alternative complement pathway. Spontaneous lysis was assessed by released hemoglobin, as previously described.9 (B) Flow cytometric analysis of C3 deposition on activated and late apoptotic human umbilical vein endothelial cells (HUVECs). Primary HUVECs (passage 3) were cultured and treated as previously described.1,2 Briefly, resting, activated (stimulated with TNF-a and IFNg), and late apoptotic cells were incubated with 33% patient serum or healthy human serum, diluted in M199 medium. After detachment, cells were labeled to assess C3 deposition using a monoclonal anti-C3c antibody (Quidel) and staining was detected by flow cytometry. Black and solid gray lines denote HUVECs incubated with patient and control serum, respectively. Irrelevant IgG1 was used as an isotype control (dashed gray line). (C) Apoptotic HUVECs were analyzed as in panel B, except that serum was supplemented with 0, 50, 150, or 300 mg/mL of purified CFH (Comptech) prior to being added to the cells. (D) Fast protein liquid chromatography (FPLC) eluate fractions from a heparin affinity column loaded with control or patient plasma were probed by Western blotting for the presence of CFH, using polyclonal anti-CFH antiserum (which also recognizes CFH-related proteins 1, 2, and 5. The smaller band was presumably CFHR5. (E, F) Dilutions of control and patient plasma, adjusted to ensure the same amount of CFH, were incubated
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Rigothier et al and mesangial IgA deposits were not observed. Reassessment of specimens from the previously amputated fingers and toes also found IgA and C4d deposits on small-artery walls (Fig 1D and F). Immunofixation of plasma and urine samples revealed a monoclonal IgAl. The serum IgAl monoclonal gammopathy was also detected upon retrospective immunofixation analysis of a sample taken shortly before the finger/toe amputations. Bone marrow smears and a biopsy specimen showed that 4% of cells were plasma cells, which stained positive with anti-l and anti-a antibodies. CH50, C3, C4, factor B, CFH, factor I, and CD46 (membrane cofactor protein) levels were unremarkable (Fig S2A). Genetic analyses detected no mutations in the genes coding for CFH, factor I, CD46, factor B, and C3; however, the patient was homozygosis for a complete deletion of the CFHR1/CFHR3 locus.5 Examination of the patient’s plasma detected binding of the IgA autoantibody to CFH (Fig S2B). His plasma strongly induced lysis of nonsensitized sheep erythrocytes (Fig 2A) and enhanced C3 deposition on activated or late apoptotic, but not on resting, endothelial cells (Fig 2B). In side-by-side tests of the patient’s serum and serum from a healthy control, addition of purified CFH resulted in a decrease in C3 deposition on late apoptotic cells; however, twice as much CFH had to be added to the patient’s serum to reach 50% inhibition (Fig 2C). The interaction of CFH with the model glycosaminoglycan heparin was measured by affinity chromatography of plasma from the patient or a healthy control. CFH eluted from the control plasma appeared at fractions 25 to 29, while no CFH was present in the corresponding fractions from the patient’s plasma, suggesting that CFH from the patient’s plasma does not interact with heparin (Fig 2D). The patient’s anti-CFH autoantibody did not affect the binding of plasma CFH to C3b (Fig 2E) or C3d (Fig 2F) and did not perturb the dissociation of C3 convertase, as measured by a specific hemolytic assay (Fig 2G). Plasma exchange therapy was started, and then after 1 month, corticosteroid treatment was begun. Despite an initial improvement in clinical and biological parameters, the patient experienced multiple aHUS relapses. Throughout the patient’s course, there was a clear-cut temporal association between symptoms of distal angiopathy and biological signs of aHUS. Several therapeutic strategies were used without obvious efficacy against aHUS symptoms. Concomitant with aHUS relapses, kidney function deteriorated and hemodialysis therapy was started 5 years after initial presentation. The time course of treatments and the evolution of aHUS parameters, serum IgA levels, and kidney function are summarized in Fig S3. Five months after dialysis therapy initiation, eculizumab therapy was started, in the setting of a clinical trial of eculizumab in aHUS (ClinicalTrials.gov identifier: NCT00838513).6 The effect of eculizumab was impressive, with immediate and definitive remission of clinical symptoms and biological signs of distal angiopathy and thrombotic microangiopathy. The patient reported a drastic improvement in quality of life. He remained hemodialysis dependent for a further 7 months, then received a deceased donor kidney transplant. He continued eculizumab treatment (with supplemental administration after surgery) and experienced an uneventful time course, with no relapse of distal angiopathy or aHUS.
DISCUSSION To our knowledge, this is the first case report of anti-CFH IgA monoclonal gammopathy–associated
aHUS with severe distal angiopathy. We present evidence that the anti-CFH IgAl that was detected was functionally relevant because it inhibited CFH binding to glycosaminoglycans in vitro and impaired cell-surface protection in cell-based assays. We have previously shown that anti-CFH antibodies of the IgG isotype (directed against multiple CFH epitopes located at the amino and carboxy termini) perturbed CFH-mediated cell protection, inhibited CFH interaction with C3, and caused C3 consumption.7 In the present case, the anti-CFH activity of the patient’s IgAl antibody blocked the interaction of CFH with the cell surface. In vitro, the patient’s plasma strongly induced lysis of nonsensitized sheep erythrocytes, as is also observed in plasma from patients with CFH mutations or anti-CFH IgG autoantibodies. This effect is not observed in plasma from healthy donors.8,9 The antibodies did not perturb the functional activity of CFH toward associated C3 convertase because C3 levels remained normal throughout the disease course. Moreover, an increase of C3 deposition on cultured endothelial cells was detected from the patient’s serum as compared to control serum. This result is similar to a previous report for CFHdepleted serum or to cases of C3 gene mutations.1 Pathologic evidence of local complement activation and IgA deposition was found in the small arteries of the kidney and also those of the amputated fingers and toes. We hypothesize that binding of IgAl to CFH inhibits the interaction of CFH with glycosaminoglycans on endothelial cells, leading to local complement activation. Release of the anaphylatoxins C3a and C5a could have induced vasoconstriction, inflammation, and thrombosis of large vessels, resulting in the various clinical manifestations and finally distal necrosis. This clinical case is exceptional due to the time course of the disease (late in adult life with distal angiopathy 3 years before aHUS), the unique pathophysiology linked to the properties of the monoclonal IgAl, and the favorable outcome after eculizumab therapy. Distal angiopathy or extrarenal manifestations have been rarely reported in aHUS, and always in children.10 There are published reports of 4 children who presented with gangrenous lesions of the distal phalanges of several fingers and toes after initial presentation with aHUS.11-13 In addition, 4 children who presented with large-artery stenosis and end-stage renal disease have been reported in the literature.14,15
with immobilized (E) C3b or (F) C3d. The amount of bound CFH was detected by ELISA. (G) Increasing concentrations of the patient’s plasma (samples from 2 different dates), pooled control plasma, or plasma from an individual with CFH haploinsufficiency (FH HI) were added to sheep erythrocytes with preformed C3 convertases to dissociate the C3bBb complexes. The residual convertases present on the cell surface were measured after addition of terminal complement components by degree of cell lysis (detected spectrophotometrically at 414 nm by the released hemoglobin). 4
Am J Kidney Dis. 2015;-(-):---
Anti-FH IgA With aHUS and Distal Angiopathy
Loirat et al16 described the development of stenoses of several thoracic and cerebral arteries in a child on longterm dialysis therapy with a factor B mutation, several years after the first flare of aHUS. In a case reported in 2009, hyperactivation of the C3 convertase with C3 fragment deposition at the endothelial surface was probably responsible for a proinflammatory and thrombogenic phenotype leading to small- and largeartery stenoses.2 All these cases of distal angiopathy involved children with very severe forms of aHUS and early onset of the disease; to our knowledge, no case has been reported in adults. With regard to extrarenal complications, these have been described in .20% of patients with aHUS.17 In the large series of patients with aHUS18 from an Indian registry, only 2 of 101 developed peripheral gangrene soon after presentation.11,19 Anti-CFH antibody–associated aHUS primarily affects children between 9 and 13 years old, but can also affect adults. The anti-CFH isotype is typically IgG, but Strobel et al20 reported 3 patients with antiCFH IgA autoantibodies. Dragon-Durey et al17 have reported 7 cases of anti-CFH antibody associated with aHUS of adult onset, but none of the patients presented with monoclonal gammopathy. In this patient, IgA monoclonal gammopathy was detected retrospectively in the patient’s serum early in the history of distal angiopathy and before aHUS onset. We postulate that monoclonal IgAl is the pathophysiologic link between distal angiopathy and aHUS through chronic local complement dysregulation.21 Due to the patient’s presentation with monoclonal gammopathy of undetermined significance with a low level of circulating IgA, we opted to treat him with eculizimab (as we would any patient with aHUS).22 The dramatic effects of eculizumab on ischemic symptoms is another strong argument for complement overactivation in the pathophysiology of distal angiopathy.23 We hypothesize that the inhibition of C5 activity inhibited the release of C5a and activation of the terminal complement pathway, thereby protecting endothelial cells and limb arteries, with disappearance of distal angiopathy symptoms. In conclusion, we present a novel observation of monoclonal IgAl with anti-CFH activity leading to severe distal angiopathy and aHUS.
ACKNOWLEDGEMENTS We thank the patient for providing informed consent to permit publication of these data. Support: This study was supported by grants from the Agence Nationale de la Recherche (ANR Genopath 2009-2012 09geno03101I) and by EU FP7 grant 2012-305608 (EURenOmics) to Dr Frémeaux-Bacchi. Financial Disclosure: Drs Delmas and Combe have received speaker fees from Alexion. The authors declare that they have no other relevant financial interests. Am J Kidney Dis. 2015;-(-):---
SUPPLEMENTARY MATERIAL Figure S1: Photographs of the lower extremities during flare. Figure S2: Analysis of complement pathway components and detection of anti-CFH IgA. Figure S3: Time course of treatments and eGFR, LDH, and serum IgA levels. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2015.03.039) is available at www.ajkd.org
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