Complement alternative pathway determines disease susceptibility and severity in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis

Activation of the alternative pathway (AP) of complement is involved in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), although the underlying molecular mechanisms are unclear. To gain insight into the role of the AP, common gene variants in CFH/CFHR1-5, CFB, C3 and MCP, and longitudinal determinations of plasma C3, C4, FH, FHR-1, FHR-2, FHR-5, FB, properdin and sC5b-9 levels were analyzed in a Spanish AAV cohort consisting of 102 patients; 54 with active AAV (active cohort) and 48 in remission not receiving immunosuppressants or dialysis therapy (remission cohort). The validation cohort consisted of 100 patients with ANCA-associated glomerulonephritis. Here, we demonstrated that common genetic variants in complement components of the AP are associated with disease susceptibility (CFB32Q/W) or severity of kidney damage in AAV (CFH-H1, CFH1H2 and ΔCFHR3/1). Plasma levels of complement components were significantly different between active and remission cohorts. In longitudinal observations, a high degree of AP activation at diagnosis was associated with worse disease outcome, while high basal FHR-1 levels and lower FH/FHR-1 ratios determined severe forms of kidney associated AAV. These genetic and plasmatic findings were confirmed in the validation cohort. Additionally, autoantibodies against FH and C3 convertase were identified in one and five active patients, respectively. Thus, our study identified key genetic and plasma components of the AP that determine disease susceptibility, prognosis, and severity in AAV. Our data also suggests that balance between FH and FHR-1 is critical and supports FHR-1 as a novel AP-specific therapeutic target in AAV. Activation of the alternative pathway (AP) of complement is involved in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), although the underlying molecular mechanisms are unclear. To gain insight into the role of the AP, common gene variants in CFH/CFHR1-5, CFB, C3 and MCP, and longitudinal determinations of plasma C3, C4, FH, FHR-1, FHR-2, FHR-5, FB, properdin and sC5b-9 levels were analyzed in a Spanish AAV cohort consisting of 102 patients; 54 with active AAV (active cohort) and 48 in remission not receiving immunosuppressants or dialysis therapy (remission cohort). The validation cohort consisted of 100 patients with ANCA-associated glomerulonephritis. Here, we demonstrated that common genetic variants in complement components of the AP are associated with disease susceptibility (CFB32Q/W) or severity of kidney damage in AAV (CFH-H1, CFH1H2 and ΔCFHR3/1). Plasma levels of complement components were significantly different between active and remission cohorts. In longitudinal observations, a high degree of AP activation at diagnosis was associated with worse disease outcome, while high basal FHR-1 levels and lower FH/FHR-1 ratios determined severe forms of kidney associated AAV. These genetic and plasmatic findings were confirmed in the validation cohort. Additionally, autoantibodies against FH and C3 convertase were identified in one and five active patients, respectively. Thus, our study identified key genetic and plasma components of the AP that determine disease susceptibility, prognosis, and severity in AAV. Our data also suggests that balance between FH and FHR-1 is critical and supports FHR-1 as a novel AP-specific therapeutic target in AAV. Lay SummaryThe activation of the complement alternative pathway (AP) has been involved in the pathogenesis of antineutrophil cytoplasmic antibody–associated vasculitis (AAV), although the underlying molecular mechanisms are unclear. Here, we have identified common genetic variants in complement genes of the AP that are associated with protection to develop AAV (CFB32Q/W) or that are associated with increased severity of the disease (deletion of CFHR1 and CFHR3, and CFH-H1). Consistent with these findings, circulating complement levels in active disease were altered compared with control and remission samples, and some of them have a prognostic value. Our data also suggest a crucial role of the balance between factor H (FH) and FH-related protein 1 in AAV. This study could explain the heterogeneity in prognosis and response to treatment observed in AAV. The activation of the complement alternative pathway (AP) has been involved in the pathogenesis of antineutrophil cytoplasmic antibody–associated vasculitis (AAV), although the underlying molecular mechanisms are unclear. Here, we have identified common genetic variants in complement genes of the AP that are associated with protection to develop AAV (CFB32Q/W) or that are associated with increased severity of the disease (deletion of CFHR1 and CFHR3, and CFH-H1). Consistent with these findings, circulating complement levels in active disease were altered compared with control and remission samples, and some of them have a prognostic value. Our data also suggest a crucial role of the balance between factor H (FH) and FH-related protein 1 in AAV. This study could explain the heterogeneity in prognosis and response to treatment observed in AAV. Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) is a systemic autoimmune disease that is characterized by necrotizing inflammation of predominantly small blood vessels and the presence of circulating ANCAs directed against myeloperoxidase or proteinase 3. The main histologic feature in the kidneys of patients with AAV is “pauci-immune” necrotizing crescentic glomerulonephritis with absent or scarce Ig and complement deposits.1Jennette J.C. Falk R.J. Hu P. Xiao H. Pathogenesis of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis.Annu Rev Pathol. 2013; 8: 139-160Crossref PubMed Scopus (200) Google Scholar,2Falk R.J. Jennette J.C. ANCA small-vessel vasculitis.J Am Soc Nephrol. 1997; 8: 314-322Crossref PubMed Google Scholar The complement system was not, therefore, initially thought to be associated with the development of AAV. Convincing evidence from animal models and clinical observations indicates, however, that activation of the complement system, the alternative pathway (AP) in particular, is pivotal for the development of AAV.3Gou S.J. Yuan J. Chen M. et al.Circulating complement activation in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.Kidney Int. 2012; 83: 129-137Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 4Villacorta J. Diaz-Crespo F. Acevedo M. et al.Glomerular C3d as a novel prognostic marker for renal vasculitis.Hum Pathol. 2016; 56: 31-39Crossref PubMed Scopus (28) Google Scholar, 5Xiao H. Schreiber A. Heeringa P. et al.Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.Am J Pathol. 2007; 170: 52-64Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar C5a is a potent anaphylatoxin and chemoattractant, crucial in disease pathogenesis,5Xiao H. Schreiber A. Heeringa P. et al.Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.Am J Pathol. 2007; 170: 52-64Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar and blockade of C5a or C5a receptor (CD88) ameliorates anti-myeloperoxidase necrotizing and crescentic glomerulonephritis in mice.6Xiao H. Dairaghi D.J. Powers J.P. et al.C5a receptor (CD88) blockade protects against MPO-ANCA GN.J Am Soc Nephrol. 2014; 25: 225-231Crossref PubMed Scopus (243) Google Scholar Recently, a phase 3 clinical trial (ADVOCATE, NCT02994927) has shown that C5a receptor inhibition with avacopan, a small antagonist, was effective in replacing high-dose glucocorticoids in patients with AAV treated with rituximab or cyclophosphamide during induction treatment.7Jayne D.R.W. Merkel P.A. Schall T.J. et al.Avacopan for the treatment of ANCA-associated vasculitis.N Engl J Med. 2021; 384: 599-609Crossref PubMed Scopus (362) Google Scholar The complement system is a major component of the innate immunity. On its activation through any of its 3 activation pathways (classical, lectin, or alternative), the complement system displays various effector functions, including induction of inflammation and cell damage. Unlike the classical and lectin pathways that need a stimulus to be triggered, the AP is always in “ready to fire” status; therefore, regulatory mechanisms are crucial to prevent uncontrolled complement activation. Factor H (FH) is the main regulator of the AP, both in fluid phase and on cellular surfaces. In certain circumstances, the regulatory activity of FH can be counteracted by FH-related proteins (FHRs; FHR1–5), other members of the FH protein family.8Lucientes-Continente L. Marquez-Tirado B. Goicoechea de Jorge E. The factor H protein family: the switchers of the complement alternative pathway.Immunol Rev. 2023; 313: 25-45Crossref PubMed Scopus (13) Google Scholar Unlike FH, the FHRs lack the complement regulatory domains of FH but share with FH similarities within the surface recognition domains. In contrast to FH, which inactivates surface-bound C3b and prevents further C3b generation and deposition (negative regulation), surface-bound FHRs promote complement activation through the AP.9Csincsi A.I. Kopp A. Zoldi M. et al.Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.J Immunol. 2015; 194: 4963-4973Crossref PubMed Scopus (65) Google Scholar, 10Csincsi A.I. Szabo Z. Banlaki Z. et al.FHR-1 binds to C-reactive protein and enhances rather than inhibits complement activation.J Immunol. 2017; 199: 292-303Crossref PubMed Scopus (32) Google Scholar, 11Goicoechea de Jorge E. Caesar J.J. Malik T.H. et al.Dimerization of complement factor H-related proteins modulates complement activation in vivo.Proc Natl Acad Sci U S A. 2013; 110: 4685-4690Crossref PubMed Scopus (205) Google Scholar, 12Hebecker M. Jozsi M. Factor H-related protein 4 activates complement by serving as a platform for the assembly of alternative pathway C3 convertase via its interaction with C3b protein.J Biol Chem. 2012; 287: 19528-19536Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Hence, the balance between FH and the FHRs may determine the outcome of AP activation/regulation and, as we previously reported for IgA nephropathy and C3 glomerulopathy, dysfunctions or alterations in the protein levels of either FH or FHRs may lead to the development of complement-mediated diseases.13Jozsi M. Tortajada A. Uzonyi B. et al.Factor H-related proteins determine complement-activating surfaces.Trends Immunol. 2015; 36: 374-384Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Although the involvement of complement in AAV pathogenesis is unquestionable, the exact molecular mechanisms leading to dysregulation of the AP are unclear. Here, we have characterized the circulating levels of various complement components and performed genetic association studies of common complement gene variants in AAV, and we investigated their contribution to disease susceptibility, severity, and prognosis in a Spanish cohort of AAV. A total of 202 adult patients with diagnosis of AAV with kidney involvement, belonging to 2 independent cohorts (discovery and validation cohorts) from 5 Spanish centers from 2016 and 2022, were included in the study. The discovery cohort consisted of 102 patients, and it was composed of 54 patients with a diagnosis of active AAV (active cohort) and 48 patients who were in remission and not receiving immunosuppressants or dialysis therapy (remission cohort). The validation cohort was composed of 100 patients with ANCA-associated glomerulonephritis. The validation cohort was mainly used to validate the genetic studies. Moreover, we gathered plasma samples from 25 age-matched controls. For the genetic studies, 389 healthy control samples of Caucasoid origin from the Spanish Registry of atypical hemolytic uremic syndrome and patients with C3 glomerulopathy (https://www.ahusc3g.es/) were used. Patients from the discovery active cohort were longitudinally followed up for 12 months after diagnosis. Clinical visits were scheduled at diagnosis, month 1, month 2, and every 3 months thereafter. Plasma and urine samples were obtained before immunosuppressive treatment was started and at every clinical visit. In the discovery remission cohort, clinical data were recorded retrospectively, and plasma and urine samples were collected when the patient was in remission. Plasma samples of 10 patients of the validation cohort were included at the time of onset and were used to validate the plasma complement determinations observed in the discovery cohort. Kidney vasculitis was defined histologically by the presence of extracapillary proliferation associated with focal glomerular necrosis and/or small-vessel vasculitis, in the absence of significant glomerular immune deposits.2Falk R.J. Jennette J.C. ANCA small-vessel vasculitis.J Am Soc Nephrol. 1997; 8: 314-322Crossref PubMed Google Scholar Renal biopsy was performed at disease onset. Patients with secondary vasculitis were excluded. Vasculitis disease activity was recorded using the Birmingham Vasculitis Activity Score (version 3).14Luqmani R.A. Bacon P.A. Moots R.J. et al.Birmingham Vasculitis Activity Score (BVAS) in systemic necrotizing vasculitis.QJM. 1994; 87: 671-678PubMed Google Scholar Disease manifestations were scored when they were attributable to active vasculitis and occurred in the previous month (new onset or worsening). Estimated glomerular filtration rate was measured using the Chronic Kidney Disease Epidemiology Collaboration creatinine formula.15Levey A.S. Stevens L.A. Schmid C.H. et al.A new equation to estimate glomerular filtration rate.Ann Intern Med. 2009; 150: 604-612Crossref PubMed Scopus (17936) Google Scholar Remission was defined by stable or decreasing serum creatinine concentration associated with significant improvement of hematuria as well as absence of extrarenal disease activity. Relapse was defined as the presence of active urine sediment and/or an increase in creatinine concentration by >30% attributable to active vasculitis after remission was achieved. The study was approved by the Research Ethics Committees of the Fundación Hospital Alcorcon, and all patients provided written informed consent. Quantitative parameters were represented as mean and SD or median and the interquartile range. Categorical variables were expressed as frequencies or percentages. Some variables were depicted in logarithmic scale. Differences of quantitative parameters between groups were assessed using the unpaired t test or Mann-Whitney test, depending on the data distribution. Pearson correlations were performed to explore lineal relationships between FH, C3, factor B (FB), and properdin. Besides, to analyze the variation of the complement components in each patient, the Wilcoxon matched-pairs signed rank test was applied. P < 0.05 was considered significant. The comparison between allele frequencies of complement genetic variants were examined by χ2 association test or Fisher exact test. Odd ratios and 95% confidence intervals were calculated. If allele frequencies were 0, the odds ratio could not be calculated and the 95% confidence interval was estimated using Cornfield correction. A Bonferroni correction for the 5 different loci studied was applied, and hence P < 0.01 was considered significant. Analyses were performed with GraphPad Prism 8 software and STATA software. Complement genetic analyses (Supplementary Figure S1), quantification of plasma (Supplementary Table S1), and urine complement components, detection of autoantibodies, quantification of urinary soluble CD163 (usCD163), and creatinine in urine are detailed in the Supplementary Methods. For the initial studies, 102 patients with a diagnosis of AAV and renal involvement were included in the study (global discovery cohort), of whom 54 patients were gathered with active disease (active cohort) and 48 patients were in remission (remission cohort). Of the patients with active AAV, 49 had newly diagnosed AAV, whereas 5 had relapsing AAV. In addition, a validation cohort consisting of 100 patients with AAV with renal involvement was used for replication studies. The clinical characteristics at the time of diagnosis of the discovery cohort (global and split active and remission patients) and of the validation cohort are listed in Table 1.Table 1Baseline clinical and histopathologic data of active and remission AAVVariableDiscovery cohortValidation cohortGlobal (n = 102)Active (n = 54)Remission (n = 48)Global (n = 100)Age at diagnosis, yr67 ± 1570 ± 1363 ± 1766 ± 13Male sex, n (%)60 (59)33 (60)27 (57)44 (44)ANCA specificity, n (%) MPO78 (76.5)46 (83.6)32 (68.1)85 (85) PR319 (18.6)9 (16.4)10 (21.3)15 (15) No ANCA5 (5)0 (0)5 (11)0 (0)Renal manifestations Histologic class, n (%)Focal19 (19)13 (24)6 (13)20 (20)Mixed30 (29)16 (29)14 (3041 (41)Crescentic19 (19)8 (15)11 (23)23 (23)Sclerotic7 (7)2 (4)5 (11)16 (16) Crescents37.4 ± 23.732.8 ± 24.143 ± 22.433 ± 24.7 Sclerosis19.3 ± 19.918.4 ± 19.120.3 ± 21.127 ± 23.0 Normal glomeruli27.1 ± 25.630.2 ± 27.323.4 ± 23.319 ± 23.0Extrarrenal manifestations, n (%)45 (44)25 (46)20 (40)50 (50)Treatment, n (%)Cyclophosphamide IV61 (60)35 (64)26 (55)43 (43)Rituximab22 (22)20 (36)2 (4)42 (42)Plasmapheresis18 (18)10 (18)8 (17)29 (29)Acute HD, n (%)27 (27)16 (29)11 (23)21 (21)Serum C3, mg/dl120 ± 25.2117 ± 25.1124 ± 25.4102 ± 47.6Serum C4, mg/dl18.6 ± 10.514.2 ± 8.3127.7 ± 8.8124.4 ± 13.0Serum creatinine, mg/dl4.00 ± 3.004.06 ± 3.004.03 ± 3.024.00 ± 3.30eGFR, ml/min per 1.73 m223.6 ± 18.922.5 ± 17.041.3 ± 42.723.0 ± 19.0Proteinuria, g/24 h0.85 [0.50–1.50]0.90 [0.50–1.80]0.80 [0.60–1.80]1.33 [0.53–2.10]BVAS index16 ± 516 ± 415 ± 516 ± 5AAV, antineutrophil cytoplasmic antibody–associated vasculitis; ANCA, antineutrophil cytoplasmic antibody; BVAS, Birmingham Vasculitis Activity Score; C3, complement C3; eGFR, estimated glomerular filtration rate; HD, hemodialysis; MPO, myeloperoxidase; PR3, proteinase 3.Data are given as mean ± SD or median [interquartile range] unless otherwise indicated. Open table in a new tab AAV, antineutrophil cytoplasmic antibody–associated vasculitis; ANCA, antineutrophil cytoplasmic antibody; BVAS, Birmingham Vasculitis Activity Score; C3, complement C3; eGFR, estimated glomerular filtration rate; HD, hemodialysis; MPO, myeloperoxidase; PR3, proteinase 3. Data are given as mean ± SD or median [interquartile range] unless otherwise indicated. To investigate if genetic variations in complement genes influence the susceptibility to develop AAV, common variants in CFH-CFHR1-5, C3, CFB, and MCP that are known to modulate the activity of the alternative pathway were analysed in the discovery cohort (n = 99, as DNA was not available in 3 patients) and in a matched control population (n = 389). Comparison of the allele frequencies observed in AAV with those observed in controls shows that the CFB32Q/W alleles are significantly decreased (odds ratio, 0.585; 95% confidence interval, 0.394–0.867; P = 0.008) in patients with AAV, whereas no significant differences are observed in CFH, C3, MCP, or ΔCFHR3/1 (Table 2). To validate these findings, the same genetic variants were analysed in the independent AAV validation cohort. As can be seen in Table 2, similar results were obtained, indicating that the CFB32Q/W alleles (odds ratio, 0.577; 95% confidence interval, 0.389–0.856; P = 0.006) are a protective factor for the development of AAV.Table 2Allele frequencies of common gene variant in the CFH-CFHR locus, MCP, CFB, and C3 in AAV and control populationsGenetic variantsControlaControls for CFH haplotypes, n = 225; deletion (CFHR3-CFHR1), n = 188; MCP haplotypes, n = 107; CFB alleles, n = 383; and C3 alleles, n = 389.Discovery cohort (n = 99)OR (95% CI)P valueValidation cohort (n = 100)OR (95% CI)P valuefrequencyfrequencyfrequencyCFH haplotypesH10.2830.3181.180 (0.819–1.699)0.400.3551.392 (0.974–1.989)0.078H20.2120.1770.798 (0.518–1.229)0.340.1650.735 (0.474–1.139)0.20H30.2100.1920.895 (0.587–1.366)0.670.2201.063 (0.708–1.597)0.76H4a0.1410.1411.007 (0.622–1.632)10.1150.795 (0.476–1.326)0.45H4b0.0670.0510.743 (0.355–1.556)0.480.0500.735 (0.335–1.539)0.48H50.0280.0301.099 (0.406–2.972)0.800.0200.718 (0.229–2.253)0.79H60.0210.0452.249 (0.879–5.755)0.120.0301.460 (0.523–4.160)0.58Deletion (CFHR3-CFHR1)0.2340.2200.910 (0.573–1.446)0.720.1700.691 (0.445–1.073)0.11MCPhaplotypesAAT0.6050.5910.945 (0.663–1.347)0.790.5700.472 (0.610–1.233)0.87GGC0.2800.2530.870 (0.586–1.293)0.550.2700.953 (0.646–1.406)0.84Other0.1160.1571.417 (0.857–2.343)0.190.1601.454 (0.883–2.395)0.15CFB allelesbCFB alleles R, Q, and W refer to the amino acids at position 32 in the protein.Q/W0.2760.1820.585 (0.394–0.867)0.0080.1800.577 (0.389–0.856)0.006C3 allelesG0.2010.1720.827 (0.549–1.244)0.420.2000.997 (0.676–1.470)1AAV, antineutrophil cytoplasmic antibody–associated vasculitis; CI, confidence interval; OR, odds ratio.Bold data indicate statistically significant differences.a Controls for CFH haplotypes, n = 225; deletion (CFHR3-CFHR1), n = 188; MCP haplotypes, n = 107; CFB alleles, n = 383; and C3 alleles, n = 389.b CFB alleles R, Q, and W refer to the amino acids at position 32 in the protein. Open table in a new tab AAV, antineutrophil cytoplasmic antibody–associated vasculitis; CI, confidence interval; OR, odds ratio. Bold data indicate statistically significant differences. Circulating levels of complement C3 and C4, FH, intact FB, properdin, and soluble C5b-9 (sC5b-9) were determined in plasma samples from both the active and remission patients from the discovery cohort and in healthy controls. In patients with active disease, the median concentrations of C3, FH, FB, and properdin were significantly decreased compared with patients in remission or controls (Figure 1; Supplementary Table S2). Conversely, sC5b-9 levels were significantly increased in active disease samples compared with remission or control samples. Moreover, a positive correlation was observed between FH and C3, FB, or properdin (Figure 2). Altogether, these data provide evidence of complement system activation through the AP and up to the terminal pathway during the active phase of the disease.Figure 2Positive correlations between circulating levels of factor H (FH), factor B (FB), complement C3, and properdin in patients with active antineutrophil cytoplasmic antibody–associated vasculitis. Correlation between (a) FH and C3, (b) FH and FB, and (c) FH and properdin. Pearson correlation test was applied. FP, factor P.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the activation of the AP may be modulated by the levels of FHR proteins, we also determined the plasma concentration of FHR-1, FHR-2, and FHR-5. As depicted in Figure 3, the levels of these proteins were significantly increased in active AAV compared with controls, and only FHR-1 levels were also different between active and remission samples. The increase in FHR levels, especially FHR-1, suggests that the balance between the AP regulator FH and the FHRs may be compromised during the acute phase of the disease. The alterations in complement levels observed in the discovery cohort were confirmed in some plasma samples from the validation cohort (Supplementary Figure S2). In addition to measuring complement levels, we also investigated the presence of autoantibodies to FH and C3 convertase in the discovery cohort. Of the 102 patients, 1 patient had low levels of anti-FH antibodies without being homozygous for the CFHR3/CFHR1 deletion. Interestingly, this patient had a severe course of the disease, did not reach remission, and required chronic kidney replacement therapy after a month from diagnosis. Antibodies to C3 convertase were only sought in patients with plasma C3 levels below the normal range (n = 23), identifying 5 patients who were positive. Notably, these antibodies did not recognize the independent components of C3 convertase (i.e., C3b and FB) and were identified in the patients with some of the lowest C3 levels (data not shown). It has been previously reported that plasma levels of FH and some of the FHRs are to some extent genetically determined. As expected, FHR-1 levels observed in the patients are highly determined by the presence of ΔCFHR3-CFHR1, being null in individuals with 2 copies of the deletion, intermediate in heterozygous individuals, and higher in individuals without the deletion (0 vs. 130 ± 55 and 250 ± 79 μg/ml, respectively). In addition, plasma FH levels were also significantly associated with the ΔCFHR3-CFHR1 genotypes, with patients homozygous for the ΔCFHR3-CFHR1 presenting higher FH levels compared with the heterozygotes or patients without the deletion (261 ± 47 vs. 216 ± 59 and 199 ± 59 μg/ml; P = 0.044 and P = 0.021, respectively). This is consistent with previously reported data showing that the ΔCFHR3-CFHR1 forms part of an extended haplotype that includes the gene encoding FH (CFH-H4), a haplotype that has been associated with elevated FH plasma levels.16Lores-Motta L. van Beek A.E. Willems E. et al.Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.Am J Hum Genet. 2021; 108: 1367-1384Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar We then investigated whether complement gene variants or plasma complement levels at diagnosis associate with kidney disease severity. To do so, patients from the AAV active cohort were stratified into 3 groups (T1, T2, and T3) based on tertiles of the urinary levels at disease onset of usCD163, a marker of macrophages that closely correlates with the kidney activity in patients with AAV.17Villacorta J. Lucientes L. Goicoechea E. et al.Urinary soluble CD163 as a biomarker of disease activity and relapse in antineutrophil cytoplasm antibody-associated glomerulonephritis.Clin Kidney J. 2021; 14: 212-219Crossref PubMed Scopus (9) Google Scholar Their clinical characteristics are summarized in Supplementary Table S4, where group T1 corresponds to patients with milder forms and group T3 to the most severe forms. Although our results should be taken with caution as only 50 to 53 samples were available for these analyses, both genetic variants and plasma levels of some components were significantly associated with kidney disease severity. Notably, CFH-H1 haplotype is significantly associated with increased renal severity, whereas CFH-H2 haplotype and ΔCFHR3/CFHR1 are associated with milder forms of kidney disease (Table 3; Supplementary Table S4). Consistent with these genetic observations, plasma FHR-1 levels are significantly lower in patients with the lowest usCD163 levels (T1), and hence the FH/FHR-1 ratio is higher (Table 4 and Figure 4a and b ). In addition, FH, properdin, and C3 levels are significantly reduced in cases with the highest usCD163 levels, suggesting that there is more complement activation in the severe forms of the disease (Table 4). In line with these observations, FHR-1 levels and the FH/FHR-1 ratio were also significantly different between patients with a less severe renal histologic phenotype (focal) and those patients with a more severe phenotype (mixed, crescentic, and sclerotic), according to the Berden classification18Berden A.E. Ferrario F. Hagen E.C. et al.Histopathologic classification of ANCA-associated glomerulonephritis.J Am Soc Nephrol. 2010; 21: 1628-1636Crossref PubMed Scopus (625) Google Scholar,19Diaz-Crespo F. Villacorta J. Acevedo M. et al.The predictive value of kidney biopsy in renal vasculitis: a multicenter cohort study.Hum Pathol. 2016; 52: 119-127Crossref PubMed Scopus (28) Google Scholar (Figure 4c and d).Table 3Allele frequencies of complement gene variants in patients with AAV stratified according to usCD163 tertilesGenetic variantsusCD163 T1 (n = 18)usCD163 T2 (n = 17)usCD163 T3 (n = 15)P valueFrequencyFrequencyFrequencyCFH haplotypesH10.1670.4710.6000.0010H20.3060.11800.0015H30.1670.1470.2330.73H4a0.1670.0590.0670.33H4b0.0560.05900.39H500.0290.0330.57H60.0830.05900.27Deletion (CFHR3-CFHR1)No0.6110.7060.8670.26Het0.2780.2940.1330.51Hom0.111000.16Alelle frequency0.2780.1180.0630.050MCPhaplotypesAAT0.5830.6470.6000.85GGC0.2220.1760.3330.33Other0.1940.1760.0670.30CFB allelesQ/W0.3330.1180.1670.068C3 allelesG0.2780.2650.1330.32AAV, antineutrophil cytoplasmic antibody–associated vasculitis; Het, heterozygous; Hom, homozygous; No, absence of the deletion; T1, tertile 1; T2, tertile 2; T3, tertile 3; usCD163, urinary soluble CD163.T1: usCD163, <379 ng/mmol; T2: usCD163, 380 to 1405 ng/mmol; T3: usCD163, >1405 ng/mmol. The χ2 association test was applied.Bold data indicate statistically significant differences. Open table in a new tab Table 4Circulating levels of complement components at diagnosis according to usCD163 tertilesComplement plasmatic levelsusCD163 T1 (n = 18)usCD163 T2 (n = 18)usCD163 T3 (n = 17)P valueaOrdinary 1-way analysis of variance test was applied, except for the FH/FHR-1 ratio, where the Kruskal-Wallis test was used.FH, μg/ml198 [173–245]160 [127–229]159 [121–197]0.014FHR-1, μg/ml180 [103–231]248 [146–340]257 [201–333]0.0027FH/FHR-1 ratio1.05 [0.80–1.45]0.73 [0.53–1.21]0.60 [0.54–0.71]0.0011FHR-2, μg/ml31.0 [17.6–38.2]30.7 [20.4–54.3]46.2 [39.0–71.3]0.0062FHR-5, μg/ml3.50 [2.05–4.18]3.86 [2.63–5.34]3.68 [2.51–4.50]0.30C5, μg/ml84.9 [66.8–107.5]72.00 [60.2–83.3]85