PRESENTED BY
GIUSEPPE REMUZZI

Presentation Summary

Written by Jasna Trbojevic-Stankovic
Reviewed by Giuseppe Remuzzi

“It’s an amazing first responder. It can lyse a bug in 30 seconds.”
John Atkinson, University School of Medicine, St. Louis
(studied complement more than 40 years)

Complement
The complement system is a critical modulator of the immune response. When disrupted, it can attack host cells and contribute to inflammatory conditions. The new insights into complement cascade activation mechanisms suggest that these proteins have more diverse roles and can cause more damage than previously thought (1). Many diseases are associated with complement activation including stroke, Alzheimer’s disease, age-related macular degeneration, asthma, myocardial infarction, Crohn’s disease, rheumatoid arthritis, membranous nephropathy, IgA nephropathy, systemic lupus erythematosus, ANCA vasculitis, etc. The kidney is particularly sensitive to complement-mediated damage, as illustrated by its involvement in atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis (MPGN), and C3 glomerulopathy (C3G). Furthermore, the most recent data suggest complement involvement in COVID-19 pathophysiology as well.

The role of complement in aHUS
aHUS is a rare subset of hemolytic uremic syndrome associated with microangiopathic hemolytic anemia and thrombocytopenia with predominant, but not exclusive, renal involvement. The annual incidence of aHUS is 0.5 to 2 cases per 1.000.000, with no sex-related difference. The disease has been strongly linked to mutations in genes encoding regulators and components of the complement system. Namely, by linkage analysis in three large families, aHUS has been mapped on an area of chromosome 1q32 including the gene for complement factor H (CFH) (2). So far, there have been 80 different mutations of these gene reported in aHUS patients, mostly located in the recognition domain. CFH regulates the alternative complement pathway in both the fluid phase and on surfaces. The CFH mutations in aHUS predominantly result in solid-phase restricted complement activation, since mutated CFH does not bind to C3b or glycosaminoglycans on endothelium and does not regulate complement on the cell surface.
Therapeutic complement inhibition has been studied and successfully applied in aHUS patients. A 2-year study by Licht et al. analyzed clinical benefits and outcomes of eculizumab treatment in this population concluding that it efficiently improves platelet count and estimated glomerular filtration rate with good tolerability and no safety concerns (3). However, the treatment cost in children is as much as 330.000 € per year, thus calling for consideration of the possible options to cut down the expense without affecting the treatment results. This issue is further complicated by the fact that there are no available specific biomarkers in plasma to monitor the effectiveness of therapy. An interesting approach to overcome this obstacle has been proposed by Noris et al. who suggested that following serum-induced C5b-9 endothelial deposits with confocal microscopy might help monitor therapeutic response, avoid drug overexposure, and save money (4). Galbusera et al. supported these results by monitoring serum-induced ex vivo C5b-9 deposition on cultured microvascular endothelium in a recently published study. The serum samples were obtained from 149 patients with either primary or secondary aHUS and, unlike the plasma sC5b-9 levels, the C5b-9 deposits on unstimulated endothelium corresponded well with clinical response to eculizumab therapy in these patients (Figure 1), (5). Last but not least, this assay might even identify asymptomatic carriers of complement gene mutations (6).

Figure 1. C5b-9 endothelial deposits and plasma levels pre and post eculizumab treatment of aHUS (4, 7)

Membranoproliferative glomerulonephritis and C3 nephropathy
MPGN is a rare form of chronic nephritis that occurs primarily in children and young adults and is characterized by diffuse proliferative lesions and widening of the capillary loops under light microscopy. Subgrouping of this condition is based on electron microscopy findings: type I is characterized by subendothelial deposits, type II by electron-dense deposits in the glomerular basement membrane, and type III by subendothelial and subepithelial deposits. All three types stain positive for complement component 3, however, immunoglobulin deposits are typically present in types I and II, but not in type II. To further add to the confusion, C3-positive but immunoglobulin-negative MPGN I and MPGN III have also been observed. Thus, a more recent classification differentiates immune complex-mediated MPGN (IC-MPGN), which is immunoglobulin-positive, and C3 glomerulopathies (C3G), encompassing dense deposit disease and C3 glomerulonephritis (C3GN), both characterized by C3 accumulation and absent or scarce immunoglobulin deposition (8). Still, these two entities share one common feature: a large majority of patients with either IC-MPGN or C3G have low serum C3 and normal C4 levels, indicating activation of the alternative complement pathway (9).
Mutations in genes encoding proteins pf the alternative pathway complement were found in both IC-MPGN and C3G. The most prevalent mutations are in the two components of the alternative pathway C3 convertase C3 and CFB (9).
In the majority of patients with C3G only antibodies against the C3 convertase, termed C3 Nephritic Factors (C3Nefs), can be found as a potential pathogenic factor. C3NeFs belong to a heterogeneous family of autoantibodies (NeFs) that stabilize the convertases complexes. C3NeFs bind to the assembled C3 convertase (C3bBb) and prevent its spontaneous and factor H mediated decay (10). Another nephritic factor has been characterized by the ability to stabilize the C5 convertase of the alternative pathway (C5NeF). C5NeFs bind to the assembled C5 convertase and prevent both its spontaneous and CFH mediated decay. Numerous studies have explored mechanisms of action of C3NeF on the C3bBb, but few have investigated the reactivity of patient IgG to C3bBb and to the C5 convertase (11).

Cluster analysis in patients with IC-MPGN and C3G
Acknowledging the limitations of the current MPGN/C3G classification, Iatropoulous et al. performed a cluster analysis in 173 patients with IC-MPGN and C3G (Figure 2). They distributed patients into four clusters, indicating the existence of four different pathogenetic patterns: clusters 1-3 included patients with fluid-phase complement activation, who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities; while cluster 4 included patients with solid-phase complement activation and normal or mildly altered serum C3, late disease onset, and poor renal survival. Patients in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits. Patients in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had fluid phase activation of the alternative pathway but C3 convertase activity predominates over C5 convertase activity as documented by mostly normal sC5b9 levels, and show very dense deposits on electron microscopy (12). The authors also devised a simple algorithm to assign patients with IC-MPGN/C3G to specific clusters (Figure 2). Such an approach may facilitate clarification of disease etiology, improve risk assessment for progression to end-stage renal disease, and pave the way for personalized treatment (12). Furthermore, unlike the standard pathohistology classification, cluster analysis exhibits prognostic value and predicts response to therapy (13). All these findings were further confirmed in a study by Garam et al. (14).

Figure 2. An algorithm to assign patients with IC-MPGN and C3 glomerulopathy to clusters (7, 12)

Coronavirus disease (COVID-19) and complement activation
There is accumulating evidence that complement activation induced by the novel coronavirus plays a major role in acute and chronic inflammation, endothelial cell dysfunction, thrombogenesis, and intravascular coagulation, and in severe cases contributes to multiple organ failure and death.
Besides the lungs, COVID-19 causes multiorgan damage, which also affects kidneys. COVID-19-associated tissue injury is a result of the inflammatory host immune response, hypercytokinemia, and aggressive inflammation that affect endothelial cells, resulting in endotheliitis and intravascular coagulation. The complement system represents the first response of the host immune system to COVID-19 infection (Figure 3).

Figure 3. Complement activation and vasculopathy mechanisms in COVID-2019 (7, 15)

Coronavirus binds to angiotensin-converting enzyme 2 (ACE2) on endothelial cells and activates the complement lectin, as well as the classical pathway, leading to C3b deposition. C3b participates in the formation of the C5 convertases that cleave C5 into the C5a and C5b-9. The terminal complement components promote vascular inflammation through multiple processes. The results of all these events are vascular injury and dysfunction, with the formation of blood clots. Based on this information, hypotheses appeared suggesting that blockade of the terminal complement pathway may represent a potential therapeutic option for the prevention and treatment of multiorgan damage in COVID-19 (15).
In vitro tests in which endothelium is exposed to the serum of COVID-19 positive patients showed C5b-9 formation. After the administration of different complement inhibitors, this process decreased (4). This effect was confirmed ex vivo after eculizumab administration as well, presenting a possible therapeutic option for these patients (Galbusera, personal communication).

References

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2. Warwicker P, Goodship TH, Donne RL, et al. Genetic studies into inherited and sporadic hemolytic uremic syndrome. Kidney Int. 1998 Apr;53(4):836-44. doi: 10.1111/j.1523-1755.1998.00824.x.

3. Licht C, Greenbaum LA, Muus P, et al. Efficacy and safety of eculizumab in atypical hemolytic uremic syndrome from 2-year extensions of phase 2 studies. Kidney Int. 2015 May;87(5):1061-73. doi: 10.1038/ki.2014.42

4. Noris M, Galbusera M, Gastoldi S, et al. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood. 2014;124(11):1715-26. doi: 10.1182/blood-2014-02-558296.

5. Galbusera M, Noris M, Gastoldi S, et al. An Ex Vivo Test of Complement Activation on Endothelium for Individualized Eculizumab Therapy in Hemolytic Uremic Syndrome. Am J Kidney Dis. 2019;74(1):56-72. doi: 10.1053/j.ajkd.2018.11.012.

6. Afshar-Kharghan V. COMPLEMENTing the diagnosis of aHUS. Blood. 2014;124(11):1699-700. doi: 10.1182/blood-2014-07-590356.

7. Remuzzi G. Complement inhibition in glomerular diseases: is it enough or too much? Presented at the 57th European Renal Association – European Dialysis Transplantation Association Congress (fully virtual), June 6, 2020. Available at the Virtual Meeting.

8. Sethi S, Fervenza FC. Membranoproliferative glomerulonephritis–a new look at an old entity. N Engl J Med. 2012;366(12):1119-31. doi: 10.1056/NEJMra1108178.

9. Pickering MC, D’Agati VD, Nester CM, et al. C3 glomerulopathy: consensus report. Kidney Int. 2013;84(6):1079-89. doi: 10.1038/ki.2013.377.

10. Iatropoulos P, Noris M, Mele C, et al. Complement gene variants determine the risk of immunoglobulin-associated MPGN and C3 glomerulopathy and predict long-term renal outcome. Mol Immunol. 2016;71:131-142. doi: 10.1016/j.molimm.2016.01.010.

11. Donadelli R, Pulieri P, Piras R, Iatropoulos P, Valoti E, Benigni A, Remuzzi G, Noris M Unraveling the Molecular Mechanisms Underlying Complement Dysregulation by Nephritic Factors in C3G and IC-MPGN. Front Immunol. 2018;9:2329. doi: 10.3389/fimmu.2018.02329. eCollection 2018.

12. Marinozzi MC, Chauvet S, Le Quintrec M, et al. C5 nephritic factors drive the biological phenotype of C3 glomerulopathies. Kidney Int. 2017;92(5):1232-1241. doi: 10.1016/j.kint.2017.04.017.

13. Iatropoulos P, Daina E, Curreri M, et al; Registry of Membranoproliferative Glomerulonephritis/C3 Glomerulopathy; Nastasi. Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN. J Am Soc Nephrol. 2018;29(1):283-294. doi: 10.1681/ASN.2017030258.

14. Cook HT, Pickering MC. Clusters Not Classifications: Making Sense of Complement-Mediated Kidney Injury. J Am Soc Nephrol. 2018;29(1):9-12. doi: 10.1681/ASN.2017111183.

15. Garam N, Prohászka Z, Szilágyi Á, et al. Validation of distinct pathogenic patterns in a cohort of membranoproliferative glomerulonephritis patients by cluster analysis. Clin Kidney J. 2019;13(2):225-234. doi: 10.1093/ckj/sfz073.

16. Noris M, Benigni A, Remuzzi G. The case of complement activation in COVID-19 multiorgan impact. Kidney Int. 2020;98(2):314-322. doi: 10.1016/j.kint.2020.05.013.

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