Elevated factor H–related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy

IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (ΔCFHR3-CFHR1). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, ΔCFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH. IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (ΔCFHR3-CFHR1). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, ΔCFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH. IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. The hallmark of IgAN is the deposition of immune complexes containing galactose-deficient IgA1 (Gd-IgA1) in the glomerular mesangium of the kidney.1Levy M. Berger J. Worldwide perspective of IgA nephropathy.Am J Kidney Dis. 1988; 12: 340-347Abstract Full Text PDF PubMed Scopus (214) Google Scholar, 2Zhao M.H. Zou W.Z. Liu G. Wang H. The changing spectrum of primary glomerular diseases within 15 years: a survey of 3331 patients in a single Chinese centre.Nephrol Dial Transplant. 2009; 24: 870-876Crossref PubMed Scopus (152) Google Scholar IgAN is a slow, progressive disease in which 15% to 40% of patients progress to chronic kidney disease (CKD) and require renal replacement therapies.3D'Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors.Am J Kidney Dis. 2000; 36: 227-237Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 4D'Amico G. Imbasciati E. Barbiano Di Belgioioso G. et al.Idiopathic IgA mesangial nephropathy. Clinical and histological study of 374 patients.Medicine (Baltimore). 1985; 64: 49-60Crossref PubMed Scopus (223) Google Scholar, 5Kobayashi Y. Tateno S. Hiki Y. Shigematsu H. IgA nephropathy: prognostic significance of proteinuria and histological alterations.Nephron. 1983; 34: 146-153Crossref PubMed Scopus (156) Google Scholar In addition to Gd-IgA1 deposition, IgAN is characterized by glomerular deposits of C3, properdin, C4 (C4d), mannose-binding lectin, and terminal complement components but not C1q.6Bene M.C. Faure G.C. Composition of mesangial deposits in IgA nephropathy: complement factors.Nephron. 1987; 46: 219Crossref PubMed Scopus (24) Google Scholar, 7Espinosa M. Ortega R. Sanchez M. et al.Association of C4d deposition with clinical outcomes in IgA nephropathy.Clin J Am Soc Nephrol. 2014; 9: 897-904Crossref PubMed Scopus (133) Google Scholar, 8Miyamoto H. Yoshioka K. Takemura T. et al.Immunohistochemical study of the membrane attack complex of complement in IgA nephropathy.Virchows Arch A Pathol Anat Histopathol. 1988; 413: 77-86Crossref PubMed Scopus (36) Google Scholar, 9Rauterberg E.W. Lieberknecht H.M. Wingen A.M. et al.Complement membrane attack (MAC) in idiopathic IgA-glomerulonephritis.Kidney Int. 1987; 31: 820-829Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 10Roos A. Rastaldi M.P. Calvaresi N. et al.Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.J Am Soc Nephrol. 2006; 17: 1724-1734Crossref PubMed Scopus (312) Google Scholar These findings support the view that the activation of the lectin pathway and alternative pathway (AP) plays a role in IgAN pathogenesis.10Roos A. Rastaldi M.P. Calvaresi N. et al.Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.J Am Soc Nephrol. 2006; 17: 1724-1734Crossref PubMed Scopus (312) Google Scholar, 11Zwirner J. Burg M. Schulze M. et al.Activated complement C3: a potentially novel predictor of progressive IgA nephropathy.Kidney Int. 1997; 51: 1257-1264Abstract Full Text PDF PubMed Scopus (95) Google Scholar Roos et al.10Roos A. Rastaldi M.P. Calvaresi N. et al.Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.J Am Soc Nephrol. 2006; 17: 1724-1734Crossref PubMed Scopus (312) Google Scholar classified IgAN patients into 2 groups according to glomerular staining for complement proteins; C4d-negative IgAN patients tended to have a more benign renal disease. Espinosa et al.7Espinosa M. Ortega R. Sanchez M. et al.Association of C4d deposition with clinical outcomes in IgA nephropathy.Clin J Am Soc Nephrol. 2014; 9: 897-904Crossref PubMed Scopus (133) Google Scholar also showed that the presence of mesangial C4d deposition could be used to identify IgAN patients with a poor long-term prognosis. Consistent with these findings, mesangial C3 deposition correlated with severe histologic lesions and worse renal outcomes,12Kim S.J. Koo H.M. Lim B.J. et al.Decreased circulating C3 levels and mesangial C3 deposition predict renal outcome in patients with IgA nephropathy.PLoS One. 2012; 7: e40495Crossref PubMed Scopus (98) Google Scholar, 13Komatsu H. Fujimoto S. Hara S. et al.Relationship between serum IgA/C3 ratio and progression of IgA nephropathy.Intern Med. 2004; 43: 1023-1028Crossref PubMed Scopus (47) Google Scholar and plasma levels of C3 activation fragments were elevated in IgAN patients with declining renal function.11Zwirner J. Burg M. Schulze M. et al.Activated complement C3: a potentially novel predictor of progressive IgA nephropathy.Kidney Int. 1997; 51: 1257-1264Abstract Full Text PDF PubMed Scopus (95) Google Scholar These data support the relevant contribution of complement activation to renal injury in IgAN patients and its potential value as a prognostic marker. The involvement of complement activation in IgAN pathogenesis is further supported by genome-wide association studies that demonstrates that the deletion of CFHR3 and CFHR1 genes (ΔCFHR3-CFHR1), which encode the complement factor H-related proteins (FHRs) FHR-3 and FHR-1, respectively, strongly protects from IgAN in multiple populations.14Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nature Genet. 2011; 43: 321-327Crossref PubMed Scopus (444) Google Scholar, 15Kiryluk K. Li Y. Sanna-Cherchi S. et al.Geographic differences in genetic susceptibility to IgA nephropathy: GWAS replication study and geospatial risk analysis.PLoS Genet. 2012; 8: e1002765Crossref PubMed Scopus (263) Google Scholar Factor H (FH) is the main regulator of AP, both in fluids and on cellular surfaces. FH is a relatively abundant plasma protein that is essential for restricting the action of complement to activating surfaces. FH binds to C3b, accelerates the decay of AP-related C3 convertase, and acts as a cofactor for the factor I-mediated proteolytic degradation of C3b.16Rodriguez de Cordoba S. Esparza-Gordillo J. Goicoechea de Jorge E. et al.The human complement factor H: functional roles, genetic variations and disease associations.Mol Immunol. 2004; 41: 355-367Crossref PubMed Scopus (490) Google Scholar The complement regulatory activities of FH on self and non-self surfaces are modulated by FHR.17Jozsi 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 (103) Google Scholar These proteins lack the complement regulatory domains of FH but have conserved FH surface recognition domains that enable competitive binding of FH to complement-activating surfaces. In contrast to FH binding to surfaces, which prevents further C3b generation and deposition (negative regulation), FHR binding competes with FH binding and enables C3b amplification to proceed unhindered. Thus, the relative levels of FH and FHRs, established by their expression or activity levels, are critical for modulating complement regulation and may determine susceptibility to complement-mediated injury.17Jozsi 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 (103) Google Scholar The capacity of FHRs to deregulate complement activation by competing with FH binding to surfaces explains the association of ΔCFHR3-CFHR1 with a lower risk of IgAN.14Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nature Genet. 2011; 43: 321-327Crossref PubMed Scopus (444) Google Scholar Interestingly, ΔCFHR3-CFHR1 is in strong linkage disequilibrium with a particular CFH haplotype associated with increased plasma FH levels,18Zhu L. Zhai Y.L. Wang F.M. et al.Variants in complement factor H and complement factor H-related protein genes, CFHR3 and CFHR1, affect complement activation in IgA nephropathy.J Am Soc Nephrol. 2015; 26: 1195-1204Crossref PubMed Scopus (97) Google Scholar further supporting that an excess of FH in relation to FHR-1 and FHR-3 accounts for the strong association of the extended CFH-CFHR3-CFHR1 haplotype with protection from IgAN. We measured the plasma levels of FH and FHR-1 proteins of IgAN patients, noncomplement-related autosomal dominant polycystic kidney disease (ADPKD) patients, and control individuals. IgAN patients with disease progression and ADPKD patients with CKD had elevated FHR-1 levels and FHR-1:FH ratios, which likely compromise FH function by competing for binding to C3 opsonized surfaces. In addition, 5 IgAN patients with progressive renal function decline had decreased FH levels because of pathogenic variants in CFH or CFI. Our data support that an impaired regulation of complement activation by FH plays a relevant role in IgAN pathogenesis. The main clinical and histologic characteristics of IgAN patients at diagnosis are shown in Table 1. Forty-nine patients (43.8%) had an estimated glomerular filtration rate (eGFR) of <60 ml/min per 1.73 m2. Positive C4d staining in renal biopsy was observed in 47 patients (44.8%).Table 1Clinical and histologic characteristics of IgA nephropathy patients (n = 112) at time of diagnosis and at the time of blood samplingVariableTime of diagnosis(mean [SD])Blood sampling(mean [SD])P valueAge (yr)33.5 (12.9)40.6 (13.2)>0.001Sex, number (%), female39 (34.8%)--Serum creatinine (mg/dl)1.5 (1.8)2 (2.2)>0.001eGFRaeGFR was calculated by the CKD-EPI equation. (ml/min per 1.73 m2)77.1 (34.7)61 (34.7)>0.001Proteinuria (g/d)1.7 (1.3)1.5 (1.2)0.1C4d+ in renal biopsy, number (%)47 (44.8%)--Oxford MEST classification of renal biopsyM1, number (%)72 (65.5%)E1, number (%)16 (14.5%)S1, number (%)58 (52.7%)T0 (<25%), number (%)65 (59.1%)T1 (25%–50%), number (%)25 (22.7%)T2 (>50%), number (%)20 (18.2%)E, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; M, mesangial hypercellularity; S, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis.a eGFR was calculated by the CKD-EPI equation. Open table in a new tab E, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; M, mesangial hypercellularity; S, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis. We performed a multiplex ligation-dependent probe amplification analysis in 106 IgAN patients, 46 ADPKD patients, and 188 control individuals. As reported in other populations, the frequency of the ΔCFHR3-CFHR1 allele in our Spanish IgAN cohort was significantly different from the control population (0.151 vs. 0.229, odds ratio = 0.60, 95% confidence interval = 0.38–0.94, P = 0.025) (Figure 1). Importantly, we found no significant differences between ADPKD patients and control individuals with respect to the frequency of the ΔCFHR3-CFHR1 allele (0.261 vs. 0.229, P = 0.58) (Figure 1). FH and FHR-1 plasma levels were measured in IgAN and ADPKD patients and control individuals, and data are presented according to their ΔCFHR3-CFHR1 genotype (Table 2, Figure 2, and Supplementary Figure S1). No significant differences in FH plasma levels were found between control individuals and IgAN or ADPKD patients, regardless of their ΔCFHR3-CFHR1 genotype (Table 2 and Figure 2a). As previously reported, there were differences in FH levels among ΔCFHR3-CFHR1 homozygotes, ΔCFHR3-CFHR1 heterozygotes, and non-carriers (Supplementary Figure S1). We also observed the expected differences in FHR-1 levels between ΔCFHR3-CFHR1 heterozygotes and non-carriers. Non-carriers had FHR-1 plasma levels that were approximately twice of those found in ΔCFHR3-CFHR1 heterozygotes. However, FHR-1 plasma levels were significantly higher in IgAN and ADPKD patients than in control individuals, regardless of their ΔCFHR3-CFHR1 genotype (Table 2 and Figure 2b). Consistent with these data, our analyses also demonstrated that the FHR-1:FH ratio increased in IgAN and ADPKD patients compared with that in control individuals (Figure 2c).Table 2Factor H (FH) and factor H-related protein 1 (FHR-1) levels and the FHR-1:FH ratio in Spanish IgA nephropathy (IgAN) and autosomal dominant polycystic kidney disease (ADPKD) patients and controlsControlsIgANADPKDnMean ± SDnMean ± SDP valuenMean ± SDP valueFH (μg/ml)NoaNo, Het, and Hom in the first column refer to the ΔCFHR3-CFHR1 genotypes.44156 ± 3975144 ± 45ns25146 ± 31nsHet24168 ± 4930184 ± 65ns18176 ± 51nsHom8176 ± 391104-3111 ± 28nsnMean ± SDNMean ± SDP valueNMean ± SDP valueFHR-1 (μg/ml)No44122 ± 2675155 ± 44<0.000125158 ± 470.0012Het2461 ± 3430116 ± 60<0.00011891 ± 350.0094Hom88 ± 21--3--nMean ± SDnMean ± SDP valuenMean ± SDP valueFHR-1:FHNo440.82 ± 0.24751.14 ± 0.35<0.0001251.1 ± 0.290.0093Het240.39 ± 0.2300.65 ± 0.270.0002180.53 ± 0.190.0235a No, Het, and Hom in the first column refer to the ΔCFHR3-CFHR1 genotypes. Open table in a new tab C4d immunofluorescence data from biopsies were available for 102 IgAN patients. An analysis of these patients confirmed previous results that demonstrated a statistically significant correlation between C4d+ biopsy and bad prognosis,7Espinosa M. Ortega R. Sanchez M. et al.Association of C4d deposition with clinical outcomes in IgA nephropathy.Clin J Am Soc Nephrol. 2014; 9: 897-904Crossref PubMed Scopus (133) Google Scholar with 64% of IgAN patients having C4d+ biopsy with disease progression (odds ratio = 3.13, 95% confidence interval = 1.20–8.19, P = 0.034). C4d+ staining and elevated FHR-1 levels are both independent variables associated with IgAN progression (Table 3). Importantly, the number of individuals who were double positive (C4d+ and elevated FHR-1 levels) was significantly increased among IgAN patients with disease progression (Table 4). A similar increase in the number of individuals who were C4d+ and exhibited an elevated FHR-1:FH ratio was observed among IgAN patients with disease progression (Supplementary Table S1).Table 3Univariate and multivariate analyses of independent factors associated with IgA nephropathy (IgAN) progressionFactorUnivariate modelMultivariate modelOR95% CIP valueOR95% CIP valueC4d+5.452.18–13.59<0.0011.590.38–6.650.520M13.321.28–8.560.0100.660.12–3.430.624E15.581.77–17.640.0023.980.76–20.880.103S115.184.82–47.760.0011.470.26–8.330.663T1–T29.533.76–24.17<0.0013.561.10–12.710.014Baseline eGFR (Δ1 ml/min per 1.73 m2)0.930.91–0.96<0.0010.930.89–0.96<0.001Baseline proteinuria (Δ1g/d)2.141.42–3.23<0.0010.750.16–3.450.710FHR-1 ≥ 140 μg/ml2.961.26–6.980.0134.641.13–19.000.033ΔCFHR3-CFHR11.730.76–3.940.1901.500.33–6.750.598E, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; FHR-1, factor H-related protein 1; M, mesangial hypercellularity; S, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis. Open table in a new tab Table 4Correlation of factor H-related protein 1 (FHR-1) levels of >175 μg/ml and C4d positivity with IgA nephropathy (IgAN) progressionIgAN progressionYES (n = 25)NO (n = 45)C4d−C4d+C4d−C4d+FHR-1 levels>175 μg/ml2771<175 μg/ml5112512IgAN progressionYESNOFHR-1 (>175 μg/ml)/C4d+YES71NO1844P = 0.002 Open table in a new tab E, endocapillary hypercellularity; eGFR, estimated glomerular filtration rate; FHR-1, factor H-related protein 1; M, mesangial hypercellularity; S, segmental glomerulosclerosis; T, tubular atrophy/interstitial fibrosis. To identify potential correlations between clinical parameters and FHR-1 levels and increased FHR-1:FH ratios, we performed a logistic regression analysis. Univariate analysis revealed a significant correlation between renal outcomes and certain baseline clinical factors (age, renal function, and proteinuria), renal biopsy features that are included in the MEST-Oxfords classification (mesangial and endocapillary hypercellularity, segmental glomerular sclerosis, and tubulointerstitial fibrosis), positive C4d staining, and FHR-1 serum levels. Multivariable analysis revealed that only a reduced eGFR at baseline, presence of tubulointerstitial fibrosis in >25% of renal parenchyma, and FHR-1 plasma levels of ≥140 μg/ml were associated with disease progression (Table 3). As shown in Table 3, FHR-1 levels of ≥140 μg/ml were significantly associated with a risk of doubling serum creatinine levels with odds ratio of 4.6. The discriminatory capacity of the model included an area under the curve of 0.93 (95% confidence interval = 0.88–0.98) with 97% sensitivity and 96% specificity. Consistent with these findings, categorizing patients according to progressive renal function decline (IgAN) or progression to CKD (ADPKD) showed that patients with disease progression exhibited significantly increased FHR-1 levels and FHR-1:FH ratios compared with those who did not have disease progression (Figure 3b and c and Supplementary Figure S1B and C). In contrast, no differences were found in FH plasma levels (Figure 3a and Supplementary Figure S2A). Twenty-one IgAN patients in our cohort presented with FH levels close to or below the cutoff value (84 μg/ml). We performed a sequencing analysis of CFH and CFI genes in these patients. Three patients (IgAN-013, IgAN-046, and IgAN-217) had CFH pathogenic variants, 1 (IgAN-072) had a CFH variant of uncertain significance, and 1 (IgAN-GL) had a likely pathogenic variant of CFI. All 5 patients were heterozygotes (see Supplementary Materials for a summary of the clinical records of these patients). Similar sequencing analyses were performed in 3 ADPKD patients with low FH levels, but no genetic variants were found in CFH and CFI genes (data not shown). In addition, the CFH gene was sequenced in all IgAN patients with CKD, but no additional pathogenic variants were identified. IgAN-013, with low FH levels (53 μg/ml), had a missense mutation that substitutes a cysteine residue at position 66 for tyrosine (c.197G>A, p.Cys66Tyr) (Figure 4a). This is a novel genetic variant that is predicted to be pathogenic. In fact, this variant is associated with decreased FH plasma levels in members of the IgAN-013 pedigree. An allele-specific FH expression analysis19Hakobyan S. Tortajada A. Harris C.L. et al.Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.Kidney Int. 2010; 78: 782-788Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar demonstrated that the CFH allele with the Cys66Tyr mutation is not expressed. Therefore, the Cys66Tyr mutation is a null mutation that results in partial FH deficiency. IgAN-046, also presenting very low FH levels (50 μg/ml), had the CFH mutation c.615T>A, which introduces a stop codon (p.Cys205*) (Figure 4b). This is a previously unknown null mutation that encodes a truncated FH protein not likely to be expressed, thus explaining the partial FH deficiency observed in this patient. IgAN-072, presenting FH levels of 76 μg/ml, had a missense mutation in the signal peptide that substitutes a valine residue at position 17 for a glycine (c.50T>G, p.Val17Gly) (Figure 4c). This genetic variation is also novel, but its functional consequence is unknown. Further analyses are required to confirm whether the p.Val17Gly mutation impairs FH secretion and causes the partial FH deficiency observed in this patient. Finally, IgAN-217, presenting with FH level of 67 μg/ml, had a novel CFH mutation (c.2933G>T, p.Trp978Leu) that is predicted to be pathogenic and is associated with decreased FH and C3 plasma levels in members of the IgAN-217 pedigree (Figure 4d). Using multiplex ligation-dependent probe amplification analysis and comparative genomic hybridization array, CFHR1 sequencing and copy number variation analysis were performed for all individuals with abnormally elevated FHR-1 levels; however, no mutations or extra copies of the CFHR1 gene were identified. IgAN is the most common primary glomerulonephritis and remains an important cause of CKD worldwide. However, the mechanisms that determine IgAN pathogenesis and the reasons for some patients progressing to CKD while others remain asymptomatic are unclear. Notably, genome-wide association studies and analyses of mesangial deposition of complement components in IgAN patients indicate a major role of complement activation in disease onset and prognosis. Because these data suggest that individuals presenting elevated FH levels relative to FHR-1 are protected from IgAN development, we investigated the correlations between clinical progression and complement genotype and FH and FHR-1 plasma levels using a well-characterized IgAN cohort (n = 112) that included 44 patients with disease progression. In addition to a cohort of 76 age- and gender-matched healthy control individuals, we included a cohort of patients with a renal disease unrelated to complement activation (n = 46 ADPKD patients, including 21 with CKD). We evaluated the risk contribution of the ΔCFHR3-CFHR1 allele and determined that ΔCFHR3-CFHR1 homozygotes were strongly protected from IgAN. We also found that FHR-1 levels are elevated in patients with CKD, regardless of whether they have IgAN or ADPKD, and there is an inverse correlation between renal function and elevated FHR-1 levels and FHR-1:FH ratios. Finally, we identified 5 patients with CFH or CFI mutations that resulted in partial FH deficiencies. Thus, our data support that low FH levels and excessive competition between FH and FHR-1, similar to C3 glomerulopathy,20Tortajada A. Yébenes H. Abarrategui-Garrido C. et al.C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation.J Clin Invest. 2013; 123: 2434-2446Crossref PubMed Scopus (150) Google Scholar, 21Goicoechea de Jorge E. Caesar J.J.E. 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 (199) Google Scholar influences IgAN development and prognosis. In agreement with previous studies of other populations,14Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nature Genet. 2011; 43: 321-327Crossref PubMed Scopus (444) Google Scholar, 15Kiryluk K. Li Y. Sanna-Cherchi S. et al.Geographic differences in genetic susceptibility to IgA nephropathy: GWAS replication study and geospatial risk analysis.PLoS Genet. 2012; 8: e1002765Crossref PubMed Scopus (263) Google Scholar we determined that the ΔCFHR3-CFHR1 allele confers strong protection from IgAN in the Spanish population. Notably, while the frequencies of ΔCFHR3-CFHR1 heterozygotes and non-carriers were similar between patients and control individuals, protection from IgAN appears to be associated with being homozygous for ΔCFHR3-CFHR1 (0.9% of IgAN patients are ΔCFHR3-CFHR1 homozygotes vs. 5.9% of healthy control individuals and 6.5% of ADPKD patients). The ΔCFHR3-CFHR1 allele identified in the 3 cohorts is in complete linkage disequilibrium with the CFH-H4 haplotype, which is associated with increased FH plasma levels.18Zhu L. Zhai Y.L. Wang F.M. et al.Variants in complement factor H and complement factor H-related protein genes, CFHR3 and CFHR1, affect complement activation in IgA nephropathy.J Am Soc Nephrol. 2015; 26: 1195-1204Crossref PubMed Scopus (97) Google Scholar, 20Tortajada A. Yébenes H. Abarrategui-Garrido C. et al.C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation.J Clin Invest. 2013; 123: 2434-2446Crossref PubMed Scopus (150) Google Scholar Our data confirm that the CFH-H4 haplotype predicts increased FH plasma levels. Indeed, FH levels in ΔCFHR3-CFHR1 heterozygotes increased compared with those in non-carriers in the control population (Table 2). Thus, the combination of increased FH levels and decreased FHR-1 levels, which characterizes this haplotype, decreases FHR-1/FH competition, enhances complement regulation by FH on the glomerular surfaces, and explains the association of this extended CFH(H4)-ΔCFHR3-CFHR1 haplotype with protection from IgAN. The idea that FHR-1/FH competition is critical in IgAN is reinforced by the observations reported in this study. Specifically, FHR-1 levels in our IgAN cohort significantly increased compared with those in control individuals, regardless of their ΔCFHR3-CFHR1 genotype (Figure 2a). Furthermore, these increased FHR-1 levels resulted in higher FHR-1:FH ratios among patients. A remarkable finding in this respect was a similar increase in FHR-1 levels and FHR-1:FH ratios in the ADPKD cohort, but without protection conferred by the ΔCFHR3-CFHR1 allele. Espinosa et al. previously reported that C4d+ biopsy is associated with disease progression in IgAN patients.7Espinosa M. Ortega R. Sanchez M. et al.Association of C4d deposition with clinical outcomes in IgA nephropathy.Clin J Am Soc Nephrol. 2014; 9: 897-904Crossref PubMed Scopus (133) Google Scholar We also observed a significant correlation between C4d+ biopsy and disease progression. These data suggest that complement activation and mesangial deposition of complement activation fragments, along with an unbalanced FHR-1:FH ratio associated with decreased renal function, exacerbates AP dysregulation, thereby leading to progressive renal function impairment. This complement activation and deposition, likely triggered by Gd-IgA1-containing immune complexes deposited in the mesangium, is consistent with the observation that mesangial C3 deposition is associated with poor renal outcome in IgAN patients.12Kim S.J. Koo H.M. Lim B.J. et al.Decreased circulating C3 levels and mesangial C3 deposition predict renal outcome in patients with IgA nephropathy.PLoS One. 2012; 7: e40495Crossref PubMed Scopus (98) Google Scholar As discussed below, C3 deposition likely contributes to the sustained competition between FH and FHR-1 because complement activated fragments such as iC3b and C3dg are better ligands for FHR-1 than for FH.20Tortajada A. Yébenes H. Abarrategui-Garr