Clinical features of C3GN and DDD
C3GN affects both sexes equally, usually in the third decade of life. Clinically, it manifests with hematuria (63–92% of cases) or less frequently with nephrotic syndrome (16–27% of cases). In general, the prognosis in C3GN is very poor with 23% patients progressing to the ESRD during a median follow-up period of 28 months [
82,
85]. DDD is a rare entity usually diagnosed in early childhood, affecting both genders equally [
62,
81]. Clinically, it presents with hematuria (76–89%) and subnephrotic proteinuria, but acute nephritic or nephrotic syndrome may also occur. In most affected individuals, it leads to ESRD (in approximately half of the patients within 10 years from diagnosis) [
81,
84]. Features of (PD), diabetes mellitus type 1, and ocular drusen can accompany DDD, but they have not been reported in C3GN [
84]. Drusen are whitish-yellow deposits of lipoproteins and complement components within the ocular Bruch’s membrane, beneath the retinal pigment epithelium, which are similar to those seen in AMD. However, in DDD, they have an earlier age of the onset, and they impair vision in approximately 10% of patients [
31]. Interestingly, no correlation between severity of renal and ocular disease has been found. PD is a condition appearing in early childhood, characterized by bilaterally symmetrical loss of subcutaneous facial and upper extremity fat. It is a result of complement activation in the adipose tissue [
81]. PD usually precedes the DDD onset, and it has been estimated that approximately 22% of PD patients develop DDD during 8 years from the diagnosis [
85].
C3G is characterized by complement dysregulation, meaning that complement may be activated spontaneously, due to acquired or congenital AP proteins abnormalities. The crucial role of AP dysfunction seems to be supported by recent reports of low plasma levels of AP components in C3G patients, such as C3, CFB, and CFH [
62]. C3 levels are more often depleted in DDD than in C3GN (59.1 vs. 39.6%, respectively) [
62,
85]. However, C3 plasma concentration within normal range does not exclude AP activation, as local (e.g., renal) C3 production and subsequent consumption may contribute to the disease pathogenesis [
25]. Moreover, studies have shown that iC3b is the main constituent of the dense deposits in the GBM. Both CFI and CFH are essential for C3b cleavage to iC3b, but the abnormalities of CFH, not CFI are responsible for the disease occurrence [
9,
83,
87]. This was demonstrated in an animal model, where CFH-deficient mice, which were also deficient in factor I, did not develop DDD but showed C3 mesangial deposition instead [
86]. Extensive complement activation in DDD was described in various animal models. A lethal renal pathology, comparable to human MPGNII, with C3 depletion in plasma and increased levels of MAC, was first diagnosed in 10 Yorkshire piglets [
87]. Later, CFH deficiency was identified as a cause of complement abnormality leading to the hereditary porcine MPGNII [
88]. In more recent studies, mice lacking CFH gene (
CFH−
/−) developed GN characterized by the capillary wall thickening, mesangial cell proliferation, mesangial and capillary staining for C3 and C9, and intramembranous dense deposits. Both
CFH−
/− and
CFH−/+ animals exhibited the evidence of AP dysregulation (low C3 levels, MAC concentration was not assessed in this study). Remarkably, the heterozygous deficiency was sufficient to impair control of C3 activation, but it did not cause renal pathology since both
CFH+/− and wild-type mice showed no histological evidence of MPGN. To confirm that the uncontrolled C3 activation causes MPGN in
CFH−
/− mice, a second mutation in
CFB gene was introduced. Since the lack of CFB hinders the formation of C3 convertase, mice deficient in both
CFH and
CFB genes did not show signs of GN [
89]. In the next study, mice deprived of both
CFH and
C5 genes developed less severe GN with significantly reduced mortality, glomerular cellularity, and slower worsening of renal function within 12 months of observation. Although C5 deficiency reduced MAC formation and prevented the formation of C5a, it did not affect the C3 deposition, which was sufficient to disrupt the permeability of the GBM, resulting in comparable levels of albuminuria in both
CFH−
/− and
C5−
/− mice [
90].
The studies performed by Ruseva et al. and Lesher et al. focused on the role of FP in glomerular injury. Since the 1970s, FP has been considered to be the only positive regulator of AP. It binds to C3bBb and stabilizes it thus counteracting CFH. Based on this knowledge, it has been expected that FP inhibition would ameliorate the AP activation. Conversely, enhancing FP would cause more efficient AP activation. Unexpectedly, studies on murine models, designed to inhibit FP by producing animals deprived of
CFH and
FP genes [
91] or mice lacking the
FP gene but producing small amounts of truncated
CFH (
CFHm/m; without SCRs 19-20) [
92] showed that the absence of FP exacerbated C3G. Both
CFHm/m/P−/− and
CFH−/−/
FP−/− mice developed severe GN resembling human DDD with predominant C3 depletion. Notably, there were several differences in clinical presentation between these two strains of mice. The
CFH−/−/FP−/− deficient mice presented with an increased glomerular cellularity, capillary wall thickening with double contours on LM, linear pattern of C3 deposition on IF, and subendothelial electron dense capillary deposits on EM. The
CFHm/m/FP−/− mice developed the even more severe disease since all the animals died. The renal injury in
CFHm/m/FP−/− was characterized by an intense C3 deposition along the vascular walls and slightly higher C3 plasma concentration. According to the authors, low but not entirely depleted C3 levels caused AP activation. In the absence of
FP gene and SCRs 19-20 of
CFH gene, AP activation occurred exclusively on GBM and podocytes, resulting in a linear pattern of C3 staining [
91,
92]. These studies emphasize the complexity of C3G pathogenesis. Namely, they imply the possibility of the contribution of more than one genetic abnormality in the development of the disease and its clinical phenotype.
It remains unclear why some patients present with DDD and the other with C3GN. Also, the exact trigger of AP dysregulation is unidentified. Some authors are seeking the answer in the frequent prevalence of monoclonal Ig deposition along with C3 and high frequency of plasma monoclonal Ig in C3G patients (approximately 31% of cases diagnosed with C3GN and 71% of DDD cases) [
93,
94]. An assumption that monoclonal Ig acts as autoantibody activating the AP has been made [
82,
83]. Using in situ hybridization, Elfituri et al. found monoclonal kappa light chain plasma cells infiltrating the glomeruli of an 11-year-old C3GN patient, who had no monoclonal Ig in plasma and normal serum and urine electrophoresis pattern. Based on these findings, the authors hypothesized that monoclonal Ig might be the direct cause of C3 deposition [
83].
Acquired AP abnormalities in C3G
Most cases of C3G are acquired and associated with different types of autoantibodies against the AP components (Table
2). The autoantibody against the C3 convertase termed C3 nephritic factor (C3NeF) is present in sera of 40% cases of C3GN and approximately 80% of DDD patients [
34,
62]. It has also been found in carriers of
CFH mutations (about 54% of cases), as well as in healthy individuals [
62,
67]. The triggers leading to the production of these autoantibodies are unknown. C3Nef activity is heterogeneous, depending on the stabilization of C3 convertase by C3Nef only (FP independent) or by both C3Nef and FP (FP-dependent C3Nef). All types of C3Nefs by binding to a neoepitope on C3bBb slow down the dissociation of Bb [
31,
95]. As a result, the time of C3 convertase activation is prolonged, and it becomes resistant to the AP-regulating factors. Clinically, low levels of circulating C3, C3 split products (C3dg/C3d), C3 deposits on IF and positive hemolytic assay are observed [
34,
68,
82]. However, the activation of MAC occurs only when C3 convertase is stabilized by both C3Nef and FP [
96,
97]. In a recent study, Marinozzi et al. [
98] have shown a more detailed insight on the association between FP and autoantibodies to AP components. The authors found IgG to C5 convertase (called C5Nef) in 49% of C3G cases and C3Nef in 68% of cases, including 39% patients positive for both C3 and C5Nefs. In this study, C3Nef correlated with low C3 plasma levels, but in most cases (62%) MAC levels remained below the normal range. The occurrence of C5Nef was related to both C3 consumption and high MAC levels. According to the authors, binding of FP to C3bBb stabilizes C5 convertase rather than C3 convertase. The prevalence of C5Nef was higher in C3GN (72%), while C3Nef was more frequent in DDD (71%), suggesting that the type of nephritic factors may determine the biological phenotype of C3G [
98]. These results are consistent with the previously described role of FP in animal models deficient in
CFH gene, where coexisting FP deficiency was associated with C3 depletion, whereas plasma C5 depletion was (at least in part) FP-dependent [
91,
92]. In
CFH−/− mice, FP also influenced the intraglomerular distribution of C3, since only these mice injected with human CFH presented C3 mesangial deposition, while
CFH−/−/P−/− mice did not show mesangial C3 staining [
91].
Table 2
Acquired factors predisposing to the AP-related glomerular diseases
| + | + | + |
anti-C3bBbC3b (C5Nef) [ 98] | + | − | − |
| − | + (LN) | − |
| + | + (LN) | −- |
| + | + | − |
| + | + | − |
| − | + | − |
| − | + | − |
Other autoantibodies such as anti-CFH, anti-C3b, and anti-CFB have been found in patients with C3G [
68,
95]. Various autoantibodies have different mechanisms of AP dysregulation. The anti-CFH antibody is directed precisely to the N-terminal end of CFH (SCR 1-4), resulting in the loss of CFH regulation in plasma [
95,
99]. C3Nef frequently coexists with anti-CFH, possibly having a synergistic effect on AP dysregulation in plasma. In a single report, anti-CFH was also associated with monoclonal Ig (35% cases in the small group of 17 patients), which is in line with previous reports of monoclonal Ig pathogenic involvement in C3G [
82,
83,
99].
Anti-CFB by binding to a neoepitope in native CFB or its activated form Bb enhances not only C3 convertase activation but also affects terminal pathway by blocking C5 convertase. Marrinozi et al. reported an association between anti-C3b/anti-CFB and infections. According to the authors, this may be explained by the similarities in coding sequences between C3 and certain pathogens (
Streptococcus epidermidis, Streptococcus capitis, Klebsiella pneumoniae, Shigella flexneri) [
95]. Altogether, these data indicate that autoantibodies alone may not be sufficient to trigger the disease onset. In most cases, additional factors such as the environmental or genetic abnormalities or the combination of all causes mentioned above may lead to the development of the disease. Undoubtedly, it is essential to investigate the potential risk factors further, as it may be crucial for future diagnostic and treatment recommendations.
Hereditary AP abnormalities in C3G
Although less common, genetic defects of the AP components, alone or in combination with the other, currently not well-defined factors, may contribute to C3GN development. McLean et al. [
52] in a small study (only nine patients) reported a higher frequency of a C3F allele in patients diagnosed with MPGN II. Finn et al. supported this observation in a study of 26 cases with presence of C3NeF and MPGN II or PD, where the prevalence of C3F allele was significantly higher [
51]. Recently, in one familial case of DDD, a heterozygous pathogenic variant of the
C3 gene coded a mutant C3 (ΔDG3923) protein, which was resistant to cleavage by the C3 convertase. This led to a constant production and consumption of C3 coded by normal allele and subsequent alternative pathway dysregulation in a fluid phase [
100]. However, in a single report of a familial C3GN in the French cohort, a cell surface AP dysregulation was described. The two affected individuals carried the mutation in
C3 gene (p.I734T), located in the binding region for CFH and CR1, which caused the functional deficiency of CR1 and CFH. The authors speculate that an impairment of the surface-bound AP regulation may be associated with a C3GN phenotype, whereas fluid-phase dysregulation with DDD presentation [
61]. Chen et al. reported another heterozygous mutation in the
C3 gene (c.C1774T/p.R592W) in a patient with C3GN combined with TMA and subacute tubulointerstitial nephritis. Interestingly, the patient’s brother who was not the carrier of this mutation developed atypical hemolytic syndrome (aHUS) [
101]. In a recent study, performed by Iatropoulos et al. on a group of 71 C3G cases, mutations in the AP components (
C3, CFI, CFH, and
MCP) were significantly more common in a C3G group than in controls. Fifty percent of all reported genetic abnormalities were located in the regulatory domains of the
C3 gene and included the following mutations: V619 M, R1042Q, R1303H, R13200Q, C1518R, and D1625H. A novel mutation in the
thrombomodulin gene (THBD A473 V; rs1042579) was also described. Though the authors underlined that the cumulative effect of the homozygous risk variants V62I in
CFH (rs800292) and A473 in
THBD (found in 58% of C3G patients) together with the presence of one of the above-mentioned mutations in
C3 gene, but not the single
C3 variation alone, was associated with a significant risk for C3G development [
64].
An association between an SNP in the
MCP gene (− 652 A > G; rs2796267) and C3G was reported by Servais et al. The authors also described a risk haplotype of combined
MCP mutations (−652A, −366A, IVS9-78G, IVS12 + 638G, c.2232T) [
62].
As mentioned earlier, since the GBM lacks membrane-bound AP regulators, it depends exclusively on CFH inhibiting properties, by binding and absorbing CFH. It is consistent with the fact that some
CFH mutations confer risk for DDD. A well-known mutation in
CFH gene, associated with AMD, (c.1204T > C; Y402H; rs1061170), was also reported in DDD patients by Abrera-Abeleda et al. [
34]. The protective variant (Y402Y) of rs1061170 bound with a stronger intensity to CRP and heparin than the risk variants (H402Y and H402H). However, there was no difference in binding to C3b between these three genotypes [
66]. Later, this observation was confirmed by Servais et al. [
62] in a group of 27 patients. Sethi et al. [
68] in a study of small group (5 C3G patients, including 1 with DDD, 3 with C3GN, and 1 case with microscopic features of both diseases) confirmed the AP activation (measured by MAC deposits in glomeruli) and prevalence of at least one copy of H402 allele in both DDD and C3 GN patients. Other specific allele variants in the
CFH gene (V62I/rs800292, IVS 2-18insTT, A473A) and in the
CFHR5 gene (P46S/rs12097550, −249T/C/rs9427661, −20T/C/rs9427662, IVS1 + 75T/A, IVS2 + 58C/T) are considered to be associated with DDD [
34]. The 62I variant of rs800292 confers protection against C3G, aHUS, and AMD, as it has a stronger ability to C3b binding and thus enhances the CFH-dependent AP inhibition both in fluids and on the cell surfaces [
63]. Conversely, the V62 variant allele of rs800292 is less efficient at inactivating C3b, and it is a susceptibility allele for C3G, aHUS and AMD [
64]. Aberera-Abeleda et al. described the additive cumulative risk of C3F/S (R102G; rs2230199) and HAV4-1+/− (P314L; rs1047286) SNPs of the
C3 gene together with the
CFH SNP Y402H (rs1061170) for the development of DDD. The presence of at least one copy of the both
C3 G102 (C3 F) and
C3 L314 (HAV4-1+) causes stronger affinity of C3 binding to CFB, while the
CFH H402 allele shows an impaired C3b binding that finally results in enhancing the C3 convertase activity and an uncontrolled consumption of the AP components [
50]. Habbig et al. presented two female siblings (12 and 7 years old) with the deletion of 224 lysine in rs796052138, located in the N-terminal end of CFH (SCR-domain 4), affected by a renal disease compatible with C3G. Both children were C3NeF positive, had low plasma C3 and CFB levels, and were successfully treated with chronic infusion of fresh frozen plasma [
102].
Several cases of the familial C3G have been described to be associated with various mutations in the
CFHR gene cluster. Gale et al. in Cypriot families reported a disease, which clinically presented with synpharyngitic hematuria, renal failure, and C3G features on biopsy. All affected individuals had an internal duplication of exons 2 and 3 in
CFHR5 gene, resulting in an expression of the mutant CFHR5 protein, with reduced affinity for the surface-bound complement. This new entity was called CFHR5 nephropathy, and it was classified as a subtype of C3G [
7,
75]. Other cases of C3G associated with mutant CFHR proteins have been described. For instance, the novel mutant multimeric CFHR1 protein that showed enhanced competition with CFH was a result of an intragenic duplication in N-terminal SCRs of
CFHR1 gene. The dysregulation of complement was present only on the certain surfaces, but (according to the authors) probably did not occur on the endothelium, explaining the C3G and not aHUS onset [
39]. Malik et al. [
76] reported a unique hybrid
CFHR3-
1 gene, which caused a familial C3G. The authors speculate that this new gene interferes with C3 regulation by CFH and CFHR5, resulting in uncontrolled C3 activation and its accumulation in the kidney. Chen et al., in familial cases of two DDD patients, discovered a chromosomal deletion in
CFHR gene cluster that led to the expression of a hybrid CFHR2-CFHR5 plasma protein. The fusion protein favored the C3 convertase formation by recruiting P and competing with CFH for binding to C3b [
40]. Xiao et al. reported another hybrid gene,
CFHR5-
CFHR2, in two relatives diagnosed with C3G. The novel hybrid CFHR5-CFHR2 protein facilitated the formation of large multimers of CFHR1, CFHR2, and CFHR5, which antagonized CFH-dependent AP regulation [
77]. Similarly, the mutant
CFHR1-
5 hybrid gene found in four family members diagnosed with C3G also exhibited the enhanced inhibition of CFH. As the evidence of both C3 and C5 dysregulation, all affected individuals had low C3 and FB levels and increased MAC and Bb plasma concentration. Also, in two patients, complete CFHR1 deficiency due to
CFHR3-1Δ was observed. Unlike the above-mentioned hybrid proteins, mutant
CFHR1-
5 was created as a result of a genetic rearrangement of 3
CFHRs genes, namely the heterozygous 147-kilobase deletion, spanning from
CFHR1 intron 4 to the 5′ region of
CFHR5 [
78]. Altogether, the exact pathogenic mechanism by which the genetic abnormalities in
CFHRs cause the disease is still unknown, but likely involves deregulation of the CS. Nevertheless, further studies are required to assess the exact role of CFHRs in the AP-mediated renal injury.
C3G versus aHUS
Finally, certain similarities, such as involvement of the AP, frequent prevalence of anti-FH antibodies, and reasonable response to the treatment with eculizumab raise a question whether C3G and aHUS are separate diseases or just the two sides of the same coin [
15,
103]. In general, mutations in the
C3 gene are uncommon in C3G, and they are more likely to be associated with DDD rather than C3 glomerulonephritis (C3GN) phenotype [
61,
62]. In contrast, genetic abnormalities in
C3 gene occur more often in aHUS [
104].
In C3G, complement dysregulation is due to CFH dysfunction in a fluid phase, resulting in a systemic AP activation and C3 fragments deposition in the kidney. Conversely, CFH surface-bound regulation is impaired in aHUS. Uncontrolled activation of complement occurs here directly on glomerular and microvascular endothelium, and it is responsible for the development of TMA and hemolysis of erythrocytes, without deposition of the AP components in glomeruli [
10,
105]. Notably, overt thrombosis is not always seen in aHUS biopsy samples. In line with this, the KDIGO 2017 consensus report suggests referring to aHUS as a ‘microangiopathy,’ with the term thrombotic being reserved for the cases with proven thrombosis. The KDIGO 2017 consensus underlines that the boundary between those diseases in some cases may be very subtle, and some patients may present with both entities [
106]. The ‘two-hit’ model for endothelial activation in TMA has been proposed, in which the endothelium can change its properties from preferential synthesis of vasodilators and antithrombotic molecules to production of vasoconstrictors and prothrombotic mediators. In the presence of pathogenic mutations and autoantibodies, this ‘primary beneficial response,’ aimed to protect the host against the aggression (e.g., infection with a pathogen) may lead to uncontrolled complement activation and excessive inflammation with microvascular thrombus formation. The local AP dysregulation on the endothelial cells seems to be crucial for aHUS pathogenesis, but the particular susceptibility of the glomerular endothelium is poorly understood. One possible explanation may lay in the heterogeneous pattern of expression of the different hemostatic mediators on various types of the endothelium (including glomerular endothelium), as well as the tissue-restricted production of distinct structures of the GAG, which can bind to CFH. Conversely, in C3G, systemic C3 activation seems to cause AP dysregulation without the involvement of the glomerular endothelium. It is not clear, why the increased production of anaphylatoxins (C3a and C5a) does not cause endothelial injury and why high levels of circulating C3b does not locally activate the AP on the endothelium in this disease [
107]. This underlies the importance of abnormalities concerning the C-terminal end of CFH protein (SCR 19-20), which in physiological conditions regulates surface-bound C3bBb, for the occurrence of aHUS (Fig.
3). In fact, autoantibodies to the C-terminal end of CFH are found in approximately 10% of aHUS patients, and the majority of them have a homozygous deletion of
CHFR1 [
37,
108,
109]. Furthermore, the prevalence of anti-CFH antibodies correlates with the occurrence of
CFHR3-
1Δ variant, known to be protective against IgAN and AMD, and a risk factor for SLE and aHUS [
47,
70,
83]. The exact impact of the same mutation being protective in some diseases and predisposing to the other remains to be specified. In case of AMD, one possible explanation is the complement deregulation caused by the absence or deficiency of either CFHR1 or CFHR3 or both these proteins [
37]. Mutations located in the N-terminal end (SCR 1-4) or autoantibodies against this part of CFH influence the C3b and C3-convertase binding in plasma and most of them are responsible for the development of C3G (Fig.
3) [
10,
25,
104]. However, in the recent studies, some N-terminal genetic abnormalities in
CFH, as well as anti-CFH antibodies to SCRs1-4, have also been reported in aHUS. Moreover, the same mutations in
CFH and
CFI have been found in both diseases, indicating that genetic abnormalities or the presence of autoantibodies are not sufficient to determine the disease phenotype. In most aHUS patients, an underlying complement abnormality is not enough to trigger the disease. The additional genetic and environmental or still unknown factors drive the patients toward one or another condition, or they remain disease-free despite the presence of gene abnormality [
62,
100,
106,
107]. The detailed role of the AP abnormalities in the pathogenesis of aHUS (due to its complexity) is currently prepared as a separate article.