C3 Glomerulopathy and Related Disorders in Children

Etiology-Phenotype Correlation and Outcomes

Abstract

Visual Abstract

Background and objectives 

Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data.

Design, setting, participants, & measurements 

A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan–Meier method.

Results 

Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2–15 (median, 9; interquartile range, 7–11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13–8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on the initial biopsy specimen.

Conclusions 

Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Introduction

Membranoproliferative GN (MPGN) is a pattern of glomerular injury characterized by increased mesangial matrix and cellularity and thickening of capillary walls (¹). MPGN classifies into immune-complex MPGN and C3 glomerulopathy on the basis of relative complement and Ig staining on biopsy specimens. C3 glomerulopathy subclassifies into dense deposit disease (DDD), with characteristic dense osmophilic intramembranous deposits, and C3 glomerulonephritis (C3GN), where other patterns of electron-dense deposition are seen (²).

Immune-complex MPGN and C3 glomerulopathy are rare, with estimated incidence of 1–4 cases per million population (³,⁴). Acquired and genetic abnormalities associated with fluid-phase dysregulation of the alternative pathway of complement have been identified in immune-complex MPGN and C3 glomerulopathy (^5–16).

Immune-complex MPGN and C3 glomerulopathy carry a poor kidney prognosis, with a median time to kidney failure of around 10 years from diagnosis (¹⁰,^17–21). After kidney transplantation, disease recurrence occurs in the majority of grafts and is the predominant cause of graft failure in 50%–90% of transplant recipients (¹⁰,¹⁹,^22–27).

A diagnosis of MPGN/C3 glomerulopathy in childhood has lifelong consequences for children and their families. Pertinent questions focus on etiology, treatment, and prognosis. Until recently, most information to address these questions is extrapolated from cohort analyses, comprising mixed groups of adults and children (¹⁰,¹¹) or small pediatric cohorts (²⁸,²⁹).

Our aim was to build a cohort of children with MPGN/C3 glomerulopathy to describe the spectrum of histologic disease, investigate the frequency of acquired and genetic alternative pathway defects, and define clear prognostic groups to facilitate counseling and stratify emerging therapeutic options in children. We extended the cohort to include patients with immune-complex GN without MPGN pattern, who did not fulfill diagnostic criteria for IgA nephropathy or systemic lupus erythematosus, whom we hypothesized may also have underlying alternative pathway dysregulation.

Here, we report our findings from the National Study of MPGN, DDD, and C3 Glomerulopathy, which recruited children from all pediatric nephrology centers in Great Britain, using the infrastructure of the National Registry of Rare Kidney Diseases (RaDaR; https://rarerenal.org/radar-registry/).

Materials and Methods

Study Design

Patients were recruited into a multicenter observational cohort study from all pediatric kidney units in Great Britain. Prevalent patients with a diagnosis of MPGN, DDD, C3GN, or immune-complex GN were identified by local clinicians and were eligible to be invited for recruitment into the study. Patients were recruited between 2011 and 2015.

Histopathologic Data

Expert central pathology review included the original light microscopy, the original biopsy specimen report, and, where available, immunostaining and electron microscopy. Kidney biopsy specimens were classified according to the C3 glomerulopathy consensus report into four different subgroups: (1) C3GN and (2) DDD (together comprising C3 glomerulopathy), and (3) immune-complex MPGN and (4) immune-complex GN (together comprising immune-complex disease [IC-disease]) (Figure 1A).

Figure 1.: Summary of pathology and age of patients included in the cohort. (A) Classification of pathology after central review. Patients with C3 glomerulopathy (C3G) were subclassified into C3 glomerulonephritis (C3GN) and dense deposit disease (DDD). Patients with non–C3 glomerulopathy had immune-complex forms of GN and were subclassified into immune-complex membranoproliferative glomerulonephritis (IC-MPGN) and immune-complex glomerulonephritis (IC-GN). (B) Age distribution of patients. Age of presentation ranged from 2 years to 15 years, categorized by pathology classification.

Clinical and Laboratory Information

Clinical data were entered into clinical record forms into the RaDaR database and included height, serum creatinine, urinary protein-creatinine ratio (P:Cr) or albumin-creatinine ratio (A:Cr), C3, and C3 and C4 nephritic factor, all collected at baseline (the time of initial diagnostic biopsy). eGFR was calculated using the modified Schwartz formula (³⁰).

UK kidney units routinely report clinical data to the Renal Registry via RaDaR—these data were extracted to provide prospective longitudinal data and determine outcomes.

Treatment Information

Details of any use of angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACE/ARB) or immunosuppression during the clinical course were extracted from RaDaR. Treatments were used at the clinicians’ discretion. In general, patients received (1) ACE/ARB use but no immunosuppression use at any time; (2) corticosteroids and no other immunosuppression; or (3) corticosteroids and mycophenolate mofetil (MMF). We identified a further group of patients receiving other nonspecific immunosuppression that we subdivided into those using azathioprine or calcineurin inhibitor and those (the intense group) who received any of cyclophosphamide, rituximab, eculizumab, or plasma exchange at any time.

Complement Testing, Autoantibodies, and Genetics

Complement and Autoantibody Testing.

At recruitment, blood samples were collected for further complement studies. Serum C3 and C4 were measured by immunoturbidimetric assays (Roche Cobas Analyzer).

Screening for C3 nephritic factor was performed by immunofixation (¹).

Screening for autoantibodies to factor H (FH) using ELISA was performed as described previously, and included epitope binding studies of short consensus repeats 1–7 (amino [N] terminus), 8–15, 16–18, and 19–20 (carboxy terminus) (²). The ELISA was adapted to screen for autoantibodies to C3b and factor B (FB) using purified proteins (Comptech) and FH-related (FHR) proteins using recombinant FHR proteins generated in mammalian cell lines. Specificity of antibodies to FHR proteins was determined by Western blotting. Screening for autoantibodies to CD35, CD46, and CD55 was performed as described previously (³).

Control samples, as indicated, were randomly selected from a batch of 200 healthy blood donors (National Health Service [NHS] Blood and Transplant), which were normally distributed and ranging in age from 17 to 72 years of age (median age was 44, 56% were female, 95% were White). The 97.5 percentile was used to assign positive results.

Complement FHR protein 5 (CFHR5) was detected by Western blotting using patient sera under nonreducing conditions. Plasma soluble C5b-9 levels were measured as described (⁴).

Genetic Screening.

Genetic screening of all exons and flanking regions of C3 (⁵), CFB (⁶), CFH (⁷), CFI (⁸), CD46 (⁹), and DGKE (¹⁰) was performed and rare genetic variants and common polymorphisms were identified after targeted next-generation sequencing and confirmatory Sanger sequencing. Rare genetic variants were defined as minor allele frequency <0.01 in the exome variant server database (evs.gs.washington.edu). Screening for genomic disorders affecting CFH, CFHR1, CFHR2, CFHR3, and CFHR5 was undertaken using multiplex ligation-dependent probe amplification (¹¹).

Definitions and Outcomes

Duration of follow-up was from baseline until latest available eGFR or kidney failure (eGFR <15 ml/min per 1.73 m², onset of maintenance dialysis, or preemptive transplantation). Patients with an eGFR of >90 ml/min per 1.73 m² at latest follow-up or those with an eGFR of <90 ml/min per 1.73 m² at latest follow-up, but their eGFR was within 15 ml/min per 1.73 m² of baseline eGFR kidney function, were classified as having either (1) complete remission if latest urinary P:Cr was <500 mg/g or equivalent, or (2) partial remission if latest urinary P:Cr was between 500 and 3000 mg/g or equivalent. Crescentic GN was defined as having crescents within >50% of viable glomeruli.

Statistical Analysis

Statistical analysis was performed using SPSS software (IBM). Baseline clinical and histologic characteristics were expressed as median (interquartile range; IQR) for continuous variables and percentage for categoric variables. These were compared using Kruskal–Wallis (for continuous variables) and Fishers exact (for categoric variables) tests. A Bonferroni correction was used for multiple comparisons. To determine risk factors for kidney survival, Cox proportional hazards models were used. We assessed baseline clinical, histologic, and complement risk factors, including complement levels at baseline and follow-up, presence of complement antibodies, and rare genetic variants. Significant risk factors for kidney survival identified by unadjusted analysis were subjected to multivariable analysis. Kidney and transplant graft survival was determined using the Kaplan–Meier method, and group comparisons were performed using the log-rank test.

The MPGN/DDD/C3 Glomerulopathy Rare Disease Group (RDG) of the Renal Association acted as a steering committee for the study.

Ethics Statement

Ethical approval for this study was granted by North Somerset and South Bristol Research Ethics Committee (reference 09/H0106/72, 11-12-09). Patients were included after informed consent/assent in accordance with the Declaration of Helsinki.

Results

Study Cohort

A total of 80 patients were recruited into the study, median 1.95 (IQR, 0.25–4.13) years from baseline and followed up for a median of 5.18 (IQR, 2.13–8.08) years. Following central histopathologic review, 39 patients were classified as having C3 glomerulopathy, including 14 patients with DDD and 25 with C3GN. The other 41 patients with IC-disease were classified as immune-complex MPGN (31 patients) and immune-complex GN (ten patients) (Figure 1A). Of the 80 patients in this study, 51 were included in the recent National Institute for Health Research (NIHR) BioResource Rare Diseases study, which reported the results of whole-genome sequencing and a genome-wide association study in 165 adult and pediatric patients with primary MPGN and C3 glomerulopathy (³¹).

Clinical Characteristics

Patients were aged 2–15 (median, 9; IQR, 7–11) years at diagnosis (Figure 1B), and 45 patients (56%) were female. Patients typically presented with nephrotic-range proteinuria (68%), hypoalbuminemia (76%), and hematuria (91%). Low eGFR (<90 ml/min per 1.73 m²) was a feature at presentation in 44% of patients. Patients with C3 glomerulopathy were the only patients to present with severe kidney dysfunction (eGFR <30 ml/min per 1.73 m²; Table 1).

Table 1. - Clinical and pathologic characteristics at presentation in pediatric C3 glomerulopathy, immune-complex membranoproliferative glomerulonephritis, and immune-complex glomerulonephritis

Characteristics	Number of Patients with Available Data	C3 Glomerulopathy		Immune-Complex Disease
		C3 Glomerulonephritis	Dense Deposit Disease	Immune-Complex Membranoproliferative Glomerulonephritis	Immune-Complex Glomerulonephritis
Number of patients	80	25	14	31	10
Age, yr, median (IQR)	80	9 (7–12)	9.5 (6–11)	9 (6–11)	8 (3–8)
Male patients, n (%)	80	8 (32)	9 (64)	14 (45)	4 (40)
Patients with nephrotic-range proteinuria (P:Cr >3000 mg/g, A:Cr >2500 mg/g, or 4+ on dipstick), n (%)	73	13 (62)	7 (54)	22 (73)	8 (89)
Patients with serum albumin ≤3.5 g/dl, n (%)	75	16 (73)	10 (71)	26 (84)	5 (63)
Patients with hematuria, n (%)	60	16 (89)	12 (100)	19 (86)	8 (100)
Patients with eGFR <90 ml/min per 1.73 m2, n (%)	75	10 (44)	12 (86)	7 (23)	4 (50)
Patients with eGFR <30 ml/min per 1.73 m2 (including patients requiring temporary KRT), n (%)	75	4 (17) a	4 (29) a	0 (0)	0 (0)
Patients with specified pattern of glomerular injury, n (% of histologic subgroup)	80
Mesangial proliferative GN		4 (16)	5 (36)	0 (0)	7 (70)
Diffuse endocapillary proliferative GN		2 (8)	0 (0)	0 (0)	2 (20)
Crescentic GN		0 (0)	4 (29)	0 (0)	0 (0)
Membranoproliferative GN		19 (76)	5 (36)	31 (100)	0 (0)
Other		0 (0)	0 (0)	0 (0)	1 (10) b
Patients with specified amount of glomerulosclerosis, n (% of histologic subgroup)	74
None		18 (79)	12 (92)	23 (82)	9 (90)
1%–25%		3 (13)	1 (8)	5 (18)	1 (10)
26%–50%		2 (9)	0 (0)	0 (0)	0 (0)
Patients with specified amount of crescents, n (% of histologic subgroup)	74
None		18 (78)	3 (23)	26 (93)	7 (70)
1%–50%		5 (22)	6 (46)	2 (7)	3 (30)
>50%		0 (0)	4 (31)	0 (0)	0 (0)
Patients with specified amount of interstitial fibrosis/tubular atrophy, n (% of histologic subgroup)	69
None		15 (71)	10 (77)	18 (69)	8 (89)
1%–25%		5 (24)	3 (23)	6 (23)	1 (11)
26%–50%		1 (5)	0 (0)	2 (8)	0 (0)

eGFR (expressed in ml/min per 1.73 m²) was calculated by modified Schwartz formula. IQR, interquartile range; P:Cr, urinary protein-creatinine ratio; A:Cr, urinary albumin-creatinine ratio.

^aIncludes one patient with C3 glomerulonephritis and three patients with dense deposit disease requiring KRT.

^bOne patient had focal and segmental necrotizing GN.

Pathologic Features

The most common pattern of glomerular injury was MPGN (55 patients; 69%), observed in 41 patients (100%) with immune-complex MPGN, five patients (36%) with DDD, and 19 patients (76%) with C3GN (Table 1). Other pathologic features are summarized in Table 1; notably, crescentic GN was observed in four patients (5%), all DDD. Most patients displayed no evidence of chronic damage.

Complement Abnormalities

Median C3 levels for the whole cohort were 0.31 (IQR, 0.14–0.50) g/L and ranged from a median of 0.15 g/L in patients with DDD to 0.50 g/L in patients with immune-complex GN. Median C4 levels for the whole cohort were 0.14 (IQR, 0.07–0.26) g/L, and levels were significantly lower in patients with immune-complex MPGN (median, 0.12 g/L) and immune-complex GN (median, 0.13 g/L) compared with patients with DDD (0.26 g/L) (P=0.02; Table 2).

Table 2. - Prevalence of complement abnormalities in C3 glomerulopathy, immune-complex membranoproliferative glomerulonephritis, and immune-complex glomerulonephritis

Complement Abnormality	Number of Patients with Available Data	C3 Glomerulopathy		Immune-Complex Disease
		C3 Glomerulonephritis	Dense Deposit Disease	Immune-Complex Membranoproliferative Glomerulonephritis	Immune-Complex Glomerulonephritis
Serum C3 at presentation (g/L), median (IQR); n	57	0.39 (0.16–0.44); 18	0.15 (0.09–0.45); 11	0.23 (0.15–0.65); 21	0.50 (0.29–0.80); 7
Serum C4 at presentation (g/L), median (IQR); n	57	0.19 (0.08–0.26); 16	0.26 (0.15–0.31); 11	0.12 (0.06–0.14); 21	0.13 (0.07–0.18); 7
Patients with C3 nephritic factor, n (%) a	80	6 (26)	8 (62)	7 (23)	1 (10)
Patients with anti-FH Ab, n (%)	78	3 (13)	3 (21)	4 (13)	3 (30)
Patients with anti-FB Ab, n (%)	77	2 (8)	1 (7)	4 (14)	0 (0)
Patients with anti-C3b Ab, n (%)	77	0 (0)	2 (15)	3 (11)	0 (0)
Patients with any complement autoantibody, n (%)	80	10 (40)	9 (64)	14 (45)	4 (40)
sC5b-9 (ng/L), median (IQR); n	72	217 (95–410); 21	232 (154–437); 12	235 (98–432); 30	225 (98–584); 9
Patients with rare genetic variant in complement gene, n (%) b	70	1 (5)	1 (8)	4 (15)	0 (0)
Patients with rare genetic variant in DGKE, n (%)	70	0 (0)	0 (0)	0 (0)	1 (13)

n refers to the number of patients with data available; percentages are expressed as number of patients tested. IQR, interquartile range; FH, factor H; Ab, autoantibody; FB, factor B; sC5b-9 soluble C5b-9; DGKE, diacyl glycerol kinase ε.

^aC3 nephritic factor detected at any point during presentation or follow-up.

^bComplement genes tested were C3, CFB, CFH, CFI, and CD46.

Autoantibodies

Autoantibodies were identified in 37 patients (46%) (Table 2); C3 nephritic factor in 22 patients (39%), autoantibodies to FH (anti-FH) in 13 patients (17%), autoantibodies to FB (anti-FB) in seven patients (9%), and autoantibodies to C3b (anti-C3b) in five patients (7%) (Figure 2). Eight patients had more than one autoantibody detected (Table 3). There were no differences in serum C3 or C4 concentration at baseline, regardless of whether an autoantibody was detected (Table 3).

Figure 2.: Screening for antibodies against complement proteins. (A) Screening serum from 78 patients with C3GN, DDD, IC-MPGN, and IC-GN for autoantibodies against complement factor H (FH). Line indicates 97.5th percentile of samples from blood donor controls, the minimum threshold for identifying an autoantibody. Screening serum from 77 patients with C3GN, DDD, IC-MPGN, and IC-GN for autoantibodies against (B) C3b and (C) complement factor B (FB). Line indicates 97.5th percentile, the minimum threshold for identifying an autoantibody. Autoantibodies were identified against C3b (five patients) and FB (seven patients). OD 450, OD measured at 450 nm; RU, response units titrated to standard published in Goodship et al. (¹⁵).

Table 3. - Complement profile of patients with anticomplement autoantibodies

Parameter	Complement Autoantibody					No Detectable Antibody	P Value a
	C3 Nephritic Factor	Anticomplement Factor H Autoantibodies	Anticomplement Factor B Autoantibodies	Anti-C3b Autoantibodies	Any Antibody
Patients testing positive, n (% of cohort)	22 (29)	13 (17)	7 (9)	5 (7)	37 (46) b	43 (54)
Serum C3 at presentation (g/L), median (IQR)	0.17 (0.09–0.50)	0.23 (0.14–0.52)	0.41 (0.09–1.15)	0.17 (0.15–0.17)	0.23 (0.13–0.69)	0.31 (0.14–0.49)	0.83
Serum C4 at presentation (g/L), median (IQR)	0.21 (0.11–0.26)	0.12 (0.09–0.26)	0.11 (0.0.08–0.26)	0.14 (0.14–0.15)	0.15 (0.06–0.26)	0.14 (0.10–0.26)	0.77
Plasma C5b9 at recruitment to study (μg/ml), median (IQR)	193 (95–360)	210 (121–998)	111 (49–339)	329 (131–416)	190 (103–342)	248 (146–466)	0.29

IQR, interquartile range.

^aP values are comparing patients from “Any Antibody” column and those in “No Detectable Antibody” column.

^bEight patients had more than one anticomplement autoantibody. All of these had a C3 nephritic factor plus additional autoantibodies as follows: one with anti–factor B, three with anti–factor H, one with anti-C3b, one with anti–factor H and anti–factor B, and two with anti–factor H and anti-C3b autoantibodies.

C3 nephritic factor was most likely to be detected in patients with DDD (62%; P=0.04; Table 2). Anti-FH bound predominantly to the N terminus of FH in ten of 13 patients and was not associated with the CFHR3/1 deletion in homozygosity (Supplemental Table 1). The age of onset of disease in this group of patients was a median of 8 (IQR, 6–9) years.

C3 levels during follow-up were lower (median, 0.55 g/L; IQR, 0.35–0.74 g/L; P=0.01) in patients who had detectable C3 nephritic factor compared with those that did not (median, 0.93 g/L; IQR, 0.69–1.08 g/L). C4 levels during follow-up were lower (median, 0.14 g/L; IQR, 0.09–0.18 g/L; P=0.03) in patients who had a detectable anti-FH antibodies compared with those that did not (median, 0.19 g/L; IQR, 0.15–0.24 g/L; Figure 3).

Figure 3.: Comparison of complement C3 and C4 levels in the cohort. Box and whisker plot showing C3 and C4 levels at diagnosis and at follow-up depending on (A and B) the four pathologic subgroups and whether patients had (C and D) detectable C3 nephritic factor or (E and F) anti–factor H (anti-FH) autoantibody.

Autoantibodies to other complement regulatory proteins (FI, CD46, CD35, CD55, and CD59) (Supplemental Figure 1) and FHR proteins (Supplemental Figure 2) were not identified. Soluble C5b-9 levels at recruitment were elevated (median, 223.3 ng/ml; IQR, 110.0–429.2 ng/ml; normal range <200 ng/ml; Supplemental Table 2), although no trends associated with presence of complement autoantibodies or rare genetic variants.

Genetic Analysis

Rare genetic variants in the complement genes examined were identified in six patients (9%; Table 2). Of these, two patients had two rare genetic variants (Supplemental Table 3). Most variants have previously been categorized as “likely benign” or of “uncertain significance” (³²,³³). Three patients with rare genetic variants (50%) also had a complement autoantibody (Supplemental Table 3).

The C3 pR102G; c304C>G single nucleotide polymorphism was associated with a higher risk of DDD (odds ratio, 3.14; 95% confidence interval [95% CI], 1.45 to 6.8; P=0.004; Supplemental Table 4). None of the other single nucleotide polymorphisms were associated with a higher risk of disease (Supplemental Table 4). No patients had the CFHR3/1 deletion in homozygosity (Supplemental Table 1). There was no evidence of other copy number variation in this cohort (data not shown) and no genomic or proteomic evidence (Supplemental Figure 3) of CFHR5 nephropathy. One previously reported patient with immune-complex GN had a likely pathogenic variant in DGKE found in homozygosity (c.323G>A; p.C108Y; Table 2) (³⁴).

Treatments

Treatments used in the cohort are summarized in Table 4 and Supplemental Table 5. Overall, 16 patients (20%) received treatment with ACE/ARB only. The remainder all received immunosuppression with at least one agent, most commonly prednisolone (22 patients) or prednisolone in combination with MMF (17 patients). Fourteen patients received a more intense regimen that included at least one of the following: rituximab, cyclophosphamide, plasma exchange, or eculizumab.

Table 4. - Treatments received in pediatric C3 glomerulopathy, immune-complex membranoproliferative glomerulonephritis, and immune-complex glomerulonephritis

Parameter	Number of Patients	Treatment
		Angiotensin- Converting Enzyme Inhibitor/ Angiotensin Receptor Blocker	Prednisolone	Prednisolone/ Mycophenolate Mofetil	Prednisolone/+	Intense
All patients, n (%)	80	16 (20)	22 (28)	17 (21)	11 (14)	14 (18)
Pathologic subgroup; n (% of subgroup) receiving each treatment
C3GN	25	7 (28)	4 (16)	3 (12)	4 (16)	7 (28)
Dense deposit disease	14	2 (14)	2 (14)	3 (21)	1 (7)	6 (43)
Immune-complex MPGN	31	4 (13)	12 (39)	8 (26)	6 (19)	1 (3)
Immune-complex GN	10	3 (30)	4 (40)	3 (30)	0 (0)	0 (0)
Patients with nephrotic-range proteinuria, n (%) a	50	8 (16)	16 (32)	13 (26)	6 (12)	7 (14)
Patients with non–nephrotic-range proteinuria, n (%)	23	7 (30)	5 (22)	3 (13)	4 (17)	4 (17)
Patients with eGFR <90 ml/min per 1.73 m2, n (%)	33	1 (3)	9 (27)	7 (21)	4 (12)	12 (36)
Patients with eGFR >90 ml/min per 1.73 m2, n (%)	42	13 (31)	12 (29)	9 (21)	6 (14)	2 (5)
Patients with serum albumin <3.5 g/dl, n (%)	57	7 (12)	15 (26)	13 (23)	9 (16)	13 (23)
Patients with serum albumin >3.5 g/dl, n (%)	18	8 (44)	5 (28)	3 (17)	1 (6)	1 (6)
Patients with histology showing >50% crescents, n (%)	4	0 (0)	0 (0)	0 (0)	0 (0)	4 (100)
Patients with histology showing <50% crescents, n (%)	76	16 (21)	22 (29)	17 (22)	11 (15)	10 (13)

eGFR (expressed in ml/min per 1.73 m²) was calculated by modified Schwartz formula. n refers to the number of patients; percentages are expressed as number of patients tested. MPGN, membranoproliferative GN; prednisolone/+, patients receiving prednisolone in combination with azathioprine or tacrolimus; intense, patients who received any of rituximab, cyclophosphamide, plasma exchange, or eculizumab; C3GN, C3 glomerulopathy; P:Cr, urinary protein-creatinine ratio; A:Cr, urinary albumin-creatinine ratio.

^aNephrotic-range proteinuria defined as P:Cr of >3000 mg/g, A:Cr of >2500 mg/g, or 4+ on dipstick.

Patients receiving ACE/ARB only were less likely to have an eGFR of <90 ml/min per 1.73 m² (P=0.002) or albumin <3.5 g/dl (P=0.006) at baseline. Patients receiving a more intense regime of immunosuppression were more likely to have C3 glomerulopathy (P=0.001), an eGFR of <90 ml/min per 1.73 m² (P<0.001), or crescentic GN (P=0.001).

Outcomes

Disease Remission.

Complete or partial remission was observed in 28 patients (71%) with C3 glomerulopathy and 36 patients (88%) with IC-disease. Among patients with C3 glomerulopathy, complete or partial remission was less likely among patients presenting with low albumin (P=0.01) or abnormal eGFR (P=0.01) and those receiving intense immunosuppression (P=0.008) (Supplemental Table 6). No clinical features were associated with a lower likelihood of remission in patients with IC-disease (Supplemental Table 7). The presence of C3 nephritic factor or anti-FH antibodies were not associated with remission in patients with either C3 glomerulopathy (Supplemental Table 6) or those with IC-disease (Supplemental Table 7).

Kidney Survival.

During the follow-up period, 11 (14%) patients had progressed to kidney failure. In a multivariable analysis that included C3 glomerulopathy, crescentic GN, glomerulosclerosis, eGFR <90 ml/min per 1.73 m² at presentation, and intense immunosuppression, only crescentic GN remained significantly associated with kidney failure (hazard ratio, 6.2; 95% CI, 1.05 to 36.8; P<0.05). The finding of rare complement gene variants, autoantibodies to complement components, or complement levels at baseline or at follow-up did not associate with progression to kidney failure.

Kidney survival according to histologic subgroup is shown in Supplemental Figure 4A. We stratified patients with C3 glomerulopathy into three groups with significantly different short- and medium-term outcomes (Supplemental Figure 4B). Of the patients with C3 glomerulopathy, all 14 patients with an eGFR of >90 ml/min per 1.73 m² at baseline did not progress to kidney failure during the course of this study. All eight patients with C3 glomerulopathy that progressed to kidney failure had an eGFR of <90 ml/min per 1.73 m² at baseline. Among these, a pattern of crescentic GN identified patients with the shortest kidney survival (mean, 1.7 years; 95% CI, 0.0 to 3.8 years) compared with those that did not have crescentic GN (mean, 8.3 years; 95% CI, 6.0 to 10.6; P=0.009). Three patients with IC-disease reached kidney failure, including two patients with immune-complex MPGN that did not progress to kidney failure until after 10 years.

Kidney Transplant.

Of 11 patients that progressed to kidney failure, eight underwent kidney transplantation. Out of nine transplant grafts, there were four cases of recurrent disease (all C3 glomerulopathy), of which two were lost due to recurrent disease (Supplemental Figure 5).

Discussion

We report comprehensive etiologic and outcome data from a national pediatric cohort of MPGN/C3 glomerulopathy.

Cohorts comprising immune-complex MPGN, DDD, and C3GN are well described (Supplemental Table 8), and the distribution of these diseases within our cohort is comparable, suggesting individual phenotypes are not seen more commonly in children. The predominant age of disease presentation in children (between 7 and 11 years) is in keeping with previous studies (²⁸).

We identified acquired alternative pathway abnormalities in approximately half of patients, including in patients with immune-complex GN, suggesting a role of complement dysregulation in immune-complex GN. Therefore, further studies are required.

C3 nephritic factor was the most commonly detected autoantibody in our cohort, although detected in a lower proportion than in previously reported mixed age-group cohorts (¹⁰,¹¹). Our lower rate of C3 nephritic factor may be due to our wide ascertainment of cases or could reflect a lower prevalence of C3 nephritic factor in children.

Anti-FH in our cohort was identified in a comparable proportion of patients to previous reports (¹⁵,¹⁶,³⁵), and we confirm specificity of anti-FH in MPGN/C3 glomerulopathy to the N-terminal regulatory domain of FH, and the lack of association with CFHR3/CFHR1 homozygous deletion in our cohort, in keeping with previous studies (¹⁵,¹⁶). We identified patients with anti-FB and anti-C3b, both previously reported in cohorts of immune-complex MPGN and C3 glomerulopathy (³⁶). The proportion with anti-FB is in keeping with the recent study showing anti-FB in 14% of patients with C3 glomerulopathy in contrast to 91% of patients with postinfectious GN (³⁷).

The rate of rare genetic variation in our cohort was low in comparison to larger cohorts (¹⁰,¹¹), and this could be due our wide ascertainment of cases or a lower rate in children compared with adults. However, the predominance of acquired abnormalities compared with genetic ones is comparable to previous cohorts (¹⁰,¹¹). A possible explanation for an autoimmune basis of MPGN/C3 glomerulopathy has been postulated in a recent study (to which 51 of our 80 patients contributed data), which showed an association of HLA type with MPGN/C3 glomerulopathy (³¹).

C3 levels were comparable, regardless of whether we identified an alternative pathway abnormality. It is possible that patients with no alternative pathway abnormality detected in our cohort have an acquired alternative pathway abnormality that we have yet to identify (e.g., C5 nephritic factor), and further work is being undertaken to assess this.

We also found that C4 levels were lower at presentation compared with at follow-up in patients with IC-disease. In previous mixed age-group cohorts, low presenting C4 was reported in up to 15% of patients (Supplemental Table 8), and in 25% of the previous largest pediatric cohort (²⁸). The finding of lower C4 may indicate a transient response to a triggering infection in pediatric MPGN/C3 glomerulopathy. In keeping with this is the observation that C4 levels were higher at recruitment to the study. C4 levels were lower at follow-up in those with anti-FH antibodies, implying ongoing classic pathway activation, possibly triggered by deposited antibody/complement immune complexes (³⁸).

In contrast, antibodies to other complement proteins were not detected (FI, CD35, CD46, CD55, and FHR proteins), and we found only one patient with a variant in DGKE (previously described in MPGN [³⁹]), suggesting these proteins do not play a major role in the etiology of MPGN/C3 glomerulopathy.

This study did not set out to determine treatment efficacy. Our data are limited to which treatments were used and are unable to take into account their timing in relation to disease onset and relationship to complement biomarkers that were performed upon recruitment to our study. However, our data help provide an overview of treatments used in children with MPGN/C3 glomerulopathy. We report favorable outcomes in a majority of patients receiving either ACE/ARB only or moderate immunosuppression, including either prednisolone only or prednisolone and MMF. The favorable outcomes of those receiving moderate immunosuppression are comparable with those in previous observational studies of children receiving prednisolone only (⁴⁰), or in mixed adult and pediatric cohorts receiving a combination of MMF and prednisolone (^41–44). Otherwise, controlled trials in immune-complex MPGN and C3 glomerulopathy are lacking, with only a randomized controlled trial in children with MPGN (before the classification of immune-complex MGPN and C3 glomerulopathy) reporting a benefit in kidney survival of long-term treatment with high-dose corticosteroids over placebo (⁴⁵); however, such doses are associated with adverse effects. In contrast, a final group (14 of 80 patients; 18%) in our cohort received intense immunosuppression. These patients predominantly had C3 glomerulopathy and were characterized by low eGFR or >50% crescents at presentation; suggesting that, at least in some patients, these clinical characteristics prompted clinicians to offer more intense immunosuppression. Despite such treatment, these patients had the poorest outcomes, highlighting that currently available treatments are likely to be ineffective in some patients with MPGN and C3 glomerulopathy and the unmet need for novel therapies.

We found that patients with C3 glomerulopathy and normal kidney function at presentation had a low risk of progression to kidney failure during follow-up. These patients and those with immune-complex MPGN appear to have a more favorable outcome than previous large cohorts (¹⁰,¹¹), and are comparable with a recently published pediatric cohort of a similar size (⁴⁰). This could reflect the wide ascertainment of our cohort, and ongoing follow-up is required to determine their longer-term risk of kidney failure. Nonetheless, these data help the clinician to offer more bespoke counseling on prognosis, possibly distinguishing patients with potentially more favorable longer-term outcomes from those with the worst short-term outcomes.

The question as to whether some children in our cohort actually had postinfectious GN, contributing to a more favorable outcome, is important. However, the vast majority of participants had evidence of ongoing kidney disease at recruitment, many months after onset, and those recruited <6 months after diagnosis did not have better outcome than those recruited >6 months after diagnosis, which points away from inadvertent inclusion of patients with postinfectious GN. Transplant recurrence rate was comparable with previously described cohorts (²⁵,²⁷).

Our study has a number of strengths. The multicenter recruitment encouraged wide ascertainment, regardless of disease severity, and minimized the bias of reporting from patients referred to a single specialist center. We were able to conduct central pathology review, which ensured consistent classification across the multiple centers, and we followed patients longitudinally through the RaDaR database. However, the study also has specific limitations not already discussed. We cannot rule out the possibility that some patients eligible for recruitment in our study were not included. Complement biomarkers from disease onset were limited to serum C3 and C4 and C3 nephritic factor and are reliant upon local assays. Data for soluble C5b-9 and other complement antibodies could only be measured from samples taken at recruitment to study, a distinct time point from baseline. Finally, our cohort had relatively few patients progressing to kidney failure, which could explain why we did not find significant associations between the complement profile of our patients and outcomes.

In summary, we propose that, in children diagnosed with MPGN/C3 glomerulopathy and immune-complex GN, a cause of alternative pathway dysregulation should be considered and that priority should be given to screening for acquired abnormalities. We would start with C3 nephritic factor and anti-FH autoantibodies, although screening for anti-FB, C3b, or rare complement genetic variants could be considered if initial screening does not identify an abnormality.

Currently available treatment strategies, including immunosuppression with a combination of MMF and corticosteroids, may have a role in management in addition to supportive treatments with ACE/ARB. However, children with abnormal kidney function at presentation, especially those with crescentic disease, should be considered a priority for studies of novel treatments, including those targeting the alternative pathway.

Disclosures

H.T. Cook reports receiving honoraria from Alexion Pharmaceuticals and having consultancy agreements with Alexion Pharmaceuticals, Apellis Pharmaceuticals, and Novartis. D.P. Gale reports attending advisory boards for Alexion Pharmaceuticals; receiving honoraria from Alexion Pharmaceuticals, Novartis, and Otsuka; having other interests in and relationships with AlportUK, as trustee, and Renal Association Rare Diseases Committee, as chair; and receiving research funding from Travere. T.H.J. Goodship reports receiving honoraria from and/or attending advisory boards for Alexion Pharmaceuticals. C.L. Harris reports receiving consultancy income from, or attending scientific advisory boards for, Biocryst Pharmaceuticals, Freeline Therapeutics, GlaxoSmithKline, Q32 Bio, and Roche (all income is donated to the university); having consultancy agreements with BioMarin Pharmaceutical, Freeline Therapeutics, Gyroscope Therapeutics, Q32 Bio Inc., and Svar Life Sciences; having patents and inventions with Cardiff University; having a secondment to Gyroscope Therapeutics; serving as a scientific advisor for, or member of, Gyroscope Therapeutics and Q32 Bio Inc; having other interests in/relationships with RaDaR via focus groups related to kidney disease; and receiving research income from Ra Pharmaceuticals. S.A. Johnson reports serving as a scientific advisor or member of the scientific advisory board for aHUS Global Registry sponsored by Alexion Pharmaceuticals (payment is made directly from Alexion to employer and is used to support research at host institution); receiving honoraria from and attending advisory boards for Alexion Pharmaceuticals; serving as a member of the grant committee for Kidney Research UK and as a trustee of the Northern Counties Kidney Research Fund charity; and attending advisory boards for Novartis Pharmaceuticals. D. Kavanagh reports receiving honoraria from Alexion Pharmaceuticals, Apellis, Gyroscope Therapeutics, Idorsia, and Novartis; receiving research funding from Alexion Pharmaceuticals and Gyroscope Therapeutics; having consultancy agreements with Alexion Pharmaceuticals, Gyroscope Therapeutics, and Novartis; attending advisory boards for Alexion Pharmaceuticals and for Novartis Pharmaceuticals; having ownership interest in, having patents and inventions with, and serving as a scientific advisor for/member of Gyroscope Therapeutics; receiving research income from Ra Pharmaceuticals; and serving as director of the UK National Renal Complement Therapeutics Centre. D. Kavanagh’s spouse works for GlaxoSmithKline. R. Malcomson reports having ownership interest (as an owner and director) in Satsuma Medical Limited, a private medicolegal pathology services limited company; undertaking fee-remunerated medicolegal work in relation to child death investigations; and receiving royalties related to editorship of medical textbooks. K.J. Marchbank reports having other interests in/relationships with aHUS UK, and RaDaR (United Kingdom) aHUS and MPGN; having consultancy agreements with Bath ASU, Catalyst Biosciences, Freeline Therapeutics, Gemini Therapeutics Ltd., and MPM Capital; receiving research funding from Catalyst Biosciences and Gemini Therapeutics; receiving honoraria from Freeline, MPM Capital, and Sanquin Research (Sanquin Blood Supply Foundation); and receiving research income from Ra Pharmaceuticals. S.D. Marks reports that the Great Ormond Street Hospital for Children NHS Foundation Trust receives funding for immunosuppressive drug studies by Astellas and Novartis and reports serving as an associate editor for pediatric transplantation for British Journal for Renal Medicine, Pediatric Nephrology, Pediatric Transplantation, and Transplantation. B.P. Morgan reports receiving honoraria from AstraZeneca; receiving research funding from Janssen; and having consultancy agreements with, and serving as a scientific advisor for or member of UCB/Ra Pharma. I.Y. Pappworth reports having consultancy agreements with K & I Consulting. M.C. Pickering reports receiving consultancy fees from Alexion Pharmaceuticals and Apellis Pharmaceuticals; having consultancy agreements with Alexion Pharmaceuticals, Apellis Pharmaceuticals, and Gyroscope Pharmaceuticals; and receiving fees for serving on the Gyroscope Pharmaceuticals Scientific Advisory Board. G. Richardson reports having consultancy agreements with AMLo Biosciences. E.K.S. Wong reports receiving honoraria from, serving as a scientific advisor or member of, and attending advisory boards for, Alexion Pharmaceuticals, BioCryst Pharmaceuticals, and Novartis Pharmaceuticals. All remaining authors have nothing to disclose.

Funding

The study was funded by Kids Kidney Research and Northern Counties Kidney Research Fund. E.K.S. Wong was funded by the Medical Research Council grant MR/K023519/1. D. Kavanagh was funded by the Wellcome Trust and the Medical Research Council. D.P. Gale is supported by St. Peter’s Trust for Kidney, Bladder and Prostate Research. M.C. Pickering is a Wellcome Trust Senior Fellow in Clinical Science, grant 212252/Z/18/Z.