Atypical Hemolytic Uremic Syndrome (aHUS) by Ana Carina Ferreira

aHUS is a complement mediated renal and rare disease, characterized by haemolytic microangiopathic anemia, thrombocytopenia, and renal impairment. Mutations in complement regulatory proteins (loss of function or even gain-of-function) seem to have a major role in this disease, that can only become evident after an additional immune insult / trigger.

ùWithout treatment, this disease leads to kidney failure, and most of these patients had recurrence of the disease in kidney grafts. Plasma exchange was the main treatment for these patients, but control of complement activation, using eculizumab, has dramatically changed the treatment of aHUS. Nevertheless, the need of lifelong treatment with this drug is an important limitation to its use, especially because it is extremely expensive. Thus, there is an urgent need for biomarkers that can help physicians to know what is the right dose and interval between doses for a given patient.


Do you know that is very important to identify the mutation implicated in the disease?

For MCP mutations (a transmembrane protein highly expressed in the kidney) we can offer a kidney transplant, knowing that recurrence is a very rare event. This is not true for other mutations.

Patients with complement factor B mutations can be resistant to plasma exchange, because it is a gain-of-function mutation, and providing fresh-frozen plasma, gives additional complement substrate.

Meet the expert - discussion of a recent paper

Noris M, Galbusera M, Gastoldi S, et al. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood 2014; 124: 1715-1726

Expert – Giuseppe Remuzzi

Vascular and glomerulus thrombosis (HE  x400).
Courtesy of Dr. Fernanda Carvalho, MD. Renal Morphology Unit – Department of Nephrology, Hospital Curry Cabral, Lisbon.

Bibliography

Complement in kidney diseases: Core curriculum 2015 – Joshua Thurmana. Am J Kidney Dis 2015; 65: 156-168

Hemolytic uremic syndrome – Marina Noris and Giuseppe Remuzzi. J Am Soc Nephrol 2005; 16: 1035-1050

Atypical hemolytic uremic syndrome – Marina Noris and Giuseppe Remuzzi. N Engl J Med 2009; 361: 1676-168

Use of eculizumab for atypical haemolytic uremic syndrome and C3 glomerulopathies – Julien Zuber, Fadi Fakhouri, Lubka Roumenina, Chantal Loirat, Veronique Frémeaux-Bacchi, French Study Group for aHUS/C3G. Nat Rev Nephrol 2012; 8: 643-657


Ana Carina Ferreira
Chair of YNP – ERA EDTA

 Comment prepared by Marina Noris and Giuseppe Remuzzi on:
“Dynamics of complement activation in aHUS and how to monitor eculizumab therapy”; Marina Noris, Miriam Galbusera, Sara Gastoldi, Paolo Macor, Federica Banterla, Elena Bresin, Claudio Tripodo, Serena Bettoni, Roberta Donadelli, Elisabetta Valoti, Francesco Tedesco, Alessandro Amore, Rosanna Coppo, Piero Ruggenenti, Eliana Gotti, and Giuseppe Remuzzi
Blood. 2014;124(11):1715-1726

Background and rationale of the study
Hemolytic uremic syndrome (HUS) is a rare disease of microangiopathic hemolysis, thrombocytopenia, and renal failure. The most common form in children is associated with infection by strains of Escherichia coli, which produce Shiga-like toxins (STEC-HUS). Approximately 5% of HUS cases in children result from infection by neuroaminidase-producing Streptococcus pneumonia (pneumococcal-HUS or neuraminidase-associated HUS). Atypical HUS (aHUS) has been used to describe those rare cases (less than 10%) in which infections by Shiga toxin-producing bacteria, or S. pneumonia, or other secondary causes can be excluded.
Diagnosis of aHUS is done by exclusion in patients with low platelet count, increased LDH, and hemolytic anemia, in which the tests for shiga-toxin producing and neuroaminidase-producing bacteria are negative and with no evidence of secondary conditions. Plasma ADAMTS13 activity should also be measured, to exclude diagnosis of thrombotic thrombocytopenic purpura (characterized by ADAMTS13 activity <10%).  When aHUS is suspected full screening for complement disease-associated abnormalities (CFH, MCP, CFI, C3, CFB, THBD mutations and CFH/CFHR rearrangements, and anti-FH autoantibodies) should be performed. However genetic studies may take several weeks to be completed, and about 40% of aHUS patients do not carry mutations in the above genes or anti-FH antibodies.
Specific and sensitive markers of complement activation in aHUS are lacking. Serum C3 and plasma sC5b-9 (soluble terminal complement complex) levels are of limited prognostic significance, since they can be normal in a large percentage of aHUS patients, even during active disease, and can be altered in conditions other than aHUS.
aHUS-associated mutant CFH, MCP, CFI, and THBD cannot fully regulate the alternative complement pathway (AP) of complement on endothelial cells. By contrast, aHUS-associated mutant proteins effectively regulate complement in fluid phase, which would explain the normal or near-normal circulating C3 levels in many mutation carriers. Gain-of-function mutations of CFB and C3 form a C3 convertase resistant to decay by endothelial cell regulators. These findings suggested that aHUS is a disease of unrestricted endothelial complement activation, which eventually causes renal microvascular thrombosis.

Methodology and novelty
Based on the above working hypothesis, we developed a new sensitive assay of complement activation at the endothelial cell level in aHUS patients. In this assay, human microvascular endothelial cells (HMEC-1 either unstimulated or pre-activated for 10 minutes with ADP) were incubated with serum from aHUS patients or from healthy subjects (control) for 4 hours. Subsequently, we quantified the amount of deposited C3 and C5b-9 on HMEC-1 by confocal microscopy. We also studied serum from unaffected patient relatives who carried complement gene mutations (mutation carriers) and unaffected relatives who did not have mutations (non-carriers). Finally we studied control patients with C3 glomerulopathies (C3G) or immune-complex-associated MPGN, which are diseases mainly characterized by fluid-phase AP activation.

Main results:
1) On unstimulated HMEC-1, serum from all patients with acute aHUS (including patients without known complement gene mutations or anti-FH antibodies), but not serum from remission, caused wider C3 and C5b-9 deposits than control serum.
2) On HMEC-1 preexposed to ADP, a condition that mimics an activated/perturbed endothelium, all acute aHUS sera but also sera from 84% and 100% of patients in remission (including patients without known complement gene mutations or anti-FH antibodies), induced excessive C3 and C5b-9 deposits, respectively. Deposits were blocked by inhibitors of the alternative pathway (AP) of complement.
3) aHUS serum-induced C5b-9 deposits did not correlate with sC5b-9 levels in the same sera and in patient plasma, indicating that endothelial C5b-9 deposits did not derive from preformed circulating C5b-9. On the same line sera from patients with C3G or IC-MPGN and high circulating sC5b-9 levels had the same effect on C5b-9 endothelial deposits as control sera.
4) Serum of unaffected mutation carriers, but not serum of non-carriers deposited more C3 and C5b-9 on ADP-activated HMEC-1 compared with control sera.
5) In eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change post-eculizumab vs. pre-treatment values, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing.

Key messages:
1) aHUS is caused by endothelial-restricted complement AP activation and increased serum-induced C5b-9 deposits on ADP-activated HMEC-1 ex vivo might be helpful to diagnose complement-associated aHUS, even in the absence of known complement gene abnormalities.
2) Clinical remission in aHUS relies on efficient endothelial complement inhibition.
3) Both in aHUS patients in remission and in their unaffected relatives who carry complement gene mutations, a trigger that will perturb the endothelium may shift the balance between complement regulation and activation toward the latter and precipitate the disease.
4) Ex vivo serum-induced endothelial C5b-9 deposits may represent a sensitive tool to monitor complement activation and eculizumab effectiveness in aHUS.

Limitations:
1) The ex vivo test can only be performed in specialized research laboratories and still needs standardization before entering in routine clinical diagnostics
2) Prospective studies in a larger number of patients are needed to prove the sensitivity of the ex vivo test to guide eculizumab dosage and spacing in aHUS patients




aHUS is a complement mediated renal and rare disease, characterized by haemolytic microangiopathic anemia, thrombocytopenia, and renal impairment. Mutations in complement regulatory proteins (loss of function or even gain-of-function) seem to have a major role in this disease, that can only become evident after an additional immune insult / trigger.

ùWithout treatment, this disease leads to kidney failure, and most of these patients had recurrence of the disease in kidney grafts. Plasma exchange was the main treatment for these patients, but control of complement activation, using eculizumab, has dramatically changed the treatment of aHUS. Nevertheless, the need of lifelong treatment with this drug is an important limitation to its use, especially because it is extremely expensive. Thus, there is an urgent need for biomarkers that can help physicians to know what is the right dose and interval between doses for a given patient.


Do you know that is very important to identify the mutation implicated in the disease?

For MCP mutations (a transmembrane protein highly expressed in the kidney) we can offer a kidney transplant, knowing that recurrence is a very rare event. This is not true for other mutations.

Patients with complement factor B mutations can be resistant to plasma exchange, because it is a gain-of-function mutation, and providing fresh-frozen plasma, gives additional complement substrate.

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