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ORIGINAL ARTICLE

Therapy and outcomes of C3 glomerulopathy and immune-complex membranoproliferative glomerulonephritis

Priyanka Khandelwal1 & Swati Bhardwaj1 & Geetika Singh2 & Aditi Sinha1 & Pankaj Hari1 & Arvind Bagga1

Received: 30 April 2020 /Revised: 13 July 2020 /Accepted: 31 July 2020 # IPNA 2020

Abstract Background Data on therapy and outcome of dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune- complex MPGN (IC-MPGN) in children are limited. Methods In this retrospective single-center study from 2007 to 2019, kidney biopsies were reviewed to include patients aged <18-years with C3 glomerulopathy and IC-MPGN. Initial immunosuppression comprised prednisolone, mycophenolate mofetil (n = 51), tacrolimus (n = 11), and/or IV cyclophosphamide (n = 20). Clinicopathological features, response to therapy, and adverse outcome (eGFRcr < 15 mL/min/1.73 m2 or death) were evaluated. Results A total of 92 patients were classified as DDD (n = 48, 52.2%), C3GN (n = 26, 28.3%), and IC-MPGN (n = 18, 19.6%) by immunohistochemistry and electron microscopy; 8 patients with DDDwere misclassified as IC-MPGN on immunofluorescence. At last follow-up (median 4.3 years), complete or partial remission occurred in 28.5, 36.1, and 16.7% patients with DDD, C3GN, and IC-MPGN, respectively. Serum albumin at onset < 2.5 g/dL (HR = 0.29, P = 0.005) and persistently low serum C3 (HR = 0.34, P = 0.02) were associated with lack of remission. The 5-year kidney survival was 62.6, 85.5, and 88.5% in patients with DDD, C3GN, and IC-MPGN, respectively (log-rank, P = 0.006). Presentation as rapidly progressive GN (HR = 11.2, P < 0.001), age > 10 years at onset (HR = 4.0, P = 0.004), and DDD (HR = 4.2, P = 0.02) were independently associated with adverse outcome; achieving remission was protective (HR = 0.04; P < 0.001). Conclusion Outcome in patients with C3 glomerulopathy and IC-MPGN was unsatisfactory, and only a small proportion of patients achieved complete or partial remission. Patients with DDDwere more likely to present with rapidly progressive GN and were at higher risk of adverse outcomes, including kidney failure.

Keywords Dense deposit disease . Rapidly progressive glomerulonephritis . Calcineurin inhibitor . Mycophenolate mofetil .

Cyclophosphamide . Children

Introduction

C 3 g l o m e r u l o p a t h y a n d i mm u n e - c o m p l e x membranoproliferative glomerulonephritis (IC-MPGN) are rare

glomerular diseases with high risk of progressive kidney failure [1, 2]. C3 glomerulopathy is classified as C3 deposition that is ≥ 2 orders ofmagnitude higher than accompanying immunoglobulin, while IC-MPGN shows predominant IgG staining on immuno- fluorescence microscopy [3–5]. C3 glomerulopathy is further classified into dense deposit disease (DDD) and C3 glomerulo- nephritis (C3GN) based on the presence or absence, respectively, of ribbon-like intramembranous electron dense deposits [4, 5]. The pathogenetic process underlying C3 glomerulopathy is dys- regulation of the alternative complement pathway [6]. C3 nephrit- ic factor (C3Nef), autoantibodies to factors H (FH), B, and C3b, and variants or copy number variations in genes regulating the alternative complement pathway may be found in patients with C3 glomerulopathy and IC-MPGN [7–9]. Therapy, especially in children, is based on case series and expert opinion and often influenced by the degree of proteinuria or kidney dysfunction

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00467-020-04736-8) contains supplementary material, which is available to authorized users.

* Pankaj Hari pankajhari@hotmail.com

1 Division of Nephrology, ICMR Center for Advanced Research in Nephrology, Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

2 Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

https://doi.org/10.1007/s00467-020-04736-8

/ Published online: 4 September 2020

Pediatric Nephrology (2021) 36:591–600

[9–13]. Immunosuppression with steroids, mycophenolate mofe- til, cyclophosphamide, calcineurin inhibitor, and rituximab has not shown consistent benefit; plasmapheresis has been used an- ecdotally [1, 2, 9–12]. While complement blockade with eculizumab might hold promise [14], availability and cost limit its use in developing countries. In the absence of therapy, long- term outcome is unsatisfactory with progression to chronic kid- ney disease (CKD) stage 5 [15]. We aimed to report the clinico- pathological features, response to therapy, and outcome in chil- dren diagnosed with C3 glomerulopathy and IC-MPGN.

Methods

A retrospective chart review of patients managed in the divi- sion of Pediatric Nephrology at this hospital fromMarch 2007 to September 2019 was performed. Patients aged < 18 years, diagnosed as types I–III MPGN, C3 glomerulopathy, or IC- MPGN on initial or re-biopsy were included following insti- tutional ethics committee approval. In addition, patients with diffuse proliferative GN with immunofluorescence (IF) show- ing glomerular staining for C3 with or without co-dominant IgG were included if complete clinical recovery did not occur within 3 months. Patients with inadequate tissue for electron microscopy (EM) or IC-MPGN secondary to hepatitis B and C or human immunodeficiency virus (HIV) were excluded. All biopsies, including those with an initial diagnosis of MPGN types I–III, were retrospectively analyzed by a renal pathologist (GS) to classify into IC-MPGN, DDD, and C3GN based on IF and EM findings (Supplementary Figs. 1 and 2). C3 glomerulopathy, defined as dominant C3 staining (inten- sity ≥ 2 orders of magnitude more than any other immunoreactant), was further classified as DDD (dense osmiophilic intramembranous deposits) or C3GN (light dense, amorphous mesangial, paramesangial, subendothelial, and subepithelial deposits) based on EM; biopsies with co- dominant or predominant IgG staining were classified as IC- MPGN [3, 13].

Clinical details

Clinical features, therapy, and outcome were recorded. Investigations included blood levels of creatinine, albumin, electrolytes, cholesterol, C3, antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), serology for hepatitis B and C and HIV, and urinalysis. The 24-h urine protein excretion was estimated at baseline and follow-up. Hypertension was defined as blood pressure > 95th centile for age, height, and sex [16]. Glomerular filtration rate (eGFRcr) was estimated using modified Schwartz equation [17]. Microscopic hematuria was defined as > 5 red blood cells per high-power field of centrifuged urine specimen. Nephrotic-range proteinuria was > 1 g/m2/day or urine-

protein-to-creatinine ratio (Up/Uc) > 2mg/mg. Nephrotic syn- drome was the presence of nephrotic range proteinuria, hypo- albuminemia (< 2.5 g/dL), and edema. Acute glomerulone- phritis (GN) was the presence of hematuria and proteinuria, with variable degrees of hypertension and acute kidney injury. Rapidly progressive GN was defined in patients with acute GN who showed rapid decline in kidney function within 7– 10 days. Chronic GN was eGFRcr < 60 mL/min/1.73 m2 with variable degrees of hematuria and proteinuria persisting for > 3 months.

Patients were followed-up every 3–6 months. Patients with eGFRcr > 30 mL/min/1.73 m2 received an angiotensin- converting enzyme inhibitor or angiotensin II receptor blocker. Immunosuppression consisted of prednisolone (60 mg/m2/day for 4 weeks, followed by 40 mg/m2 on alter- nate days for 4 weeks and tapered by 0.1–0.2 mg/kg every 2 weeks to 5 mg on alternate days) with or without mycophe- nolate mofetil (MMF; 800–1000 mg/m2/day in two divided doses) or tacrolimus (0.12–0.15 mg/kg/day; trough levels 4– 8 ng/dL). Patients with rapidly progressive GN, crescents on biopsy, or persistently deranged kidney function at onset were treated with intravenous (IV) methylprednisolone (3–6 doses of 30 mg/kg) and IV cyclophosphamide (6 doses of 500 mg/ m2 every 3–4 weeks), followed by MMF and prednisolone.

Complete remission was defined as absence of proteinuria (< 100 mg/m2/day or Up/Uc < 0.2 mg/mg), serum albumin > 3.0 g/dL and eGFRcr > 90 mL/min/1.73 m2. Partial remission was proteinuria 100–1000 mg/m2/day or Up/Uc 0.2–2.0 mg/ mg, serum albumin > 3.0 g/dL, and improved or stable eGFRcr

(± 15 mL/min/1.73 m2). Non-response was defined as lack of complete or partial remission after 6 months of therapy.

Histopathology

Biopsy specimens with at least 10 glomeruli were considered adequate. The extent of mesangial and endocapillary prolifer- ation, basement membrane thickening and splitting, propor- tion of cellular, fibrocellular, or fibrous crescents, and extent of global or focal sclerosis were noted. Interstitial fibrosis and tubular atrophy were graded (grade 0: none; grade 1: 0–25%; grade 2: 25–50%; and grade 3: > 50%). Intensity of mesangial and capillary wall staining for IgG, IgA, IgM, C3, and C1q was graded from 0 to 3+ on IF. Location and type of deposits and extent of podocyte effacement were recorded on EM.

Statistics

Data are presented as proportions and median (1st and 3rd quartiles, Q1 and Q3) and analyzed using Stata version 14.0 (Stata Corp., College Station, TX). Tests for significance in- cluded Wilcoxon signed-rank and rank-sum tests and chi- square test. Adverse outcome was defined as eGFRcr < 15 mL/min/1.73 m2 or death. Probability of kidney survival

592 Pediatr Nephrol (2021) 36:591–600

free of adverse outcome, determined using the Kaplan-Meier method, was compared in patients with DDD, C3GN, and IC- MPGN. Determinants of complete or partial remission and adverse outcome were estimated as hazards ratios, by univar- iate and multivariable analyses, using Cox proportional haz- ards model. Variables withP < 0.1 on univariate analysis were included in the multivariable models. The ratio of number of outcome events to the number of independent variables

(events per variable) in the multivariable models was ≥ 5. Two-tailed P < 0.05 was considered significant.

Results

Of 2215 initial and re-biopsies performed from 2007 to 2019, 92 biopsies were classified as C3 glomerulopathy and IC-MPGN.

Fig. 1 Flow of study showing enrollment of 92 patients from 2007 to 2019. Patients aged < 18 years, with either biopsy suggestive of membranoproliferative glomerulonephritis (MPGN) or diffuse prolifera- tive GN with dominant C3 or co-dominant IgG and persistent disease > 3 months were included. Kidney biopsies, including 18 initially diag- nosed as MPGN types I–III (7 type I, 11 type II), were reclassified as C3 glomerulopathy (n = 74) and immune-complex MPGN (n = 18). Nine patients classified as immune-complex MPGN on immunofluorescence (IF) were classified as dense deposit disease (DDD; n = 8) on electron

microscopy (EM) and C3 glomerulonephritis (C3GN; n = 1) on re-biop- sy. Four of nine misclassified patients showed predominant C3 staining on subsequent re-biopsy. aLack of enough tissue or advanced glomerulosclerosis. bInitial IF showed C3 1+ and IgG 3+ after pronase digestion; C3 3+ and absence of IgG on re-biopsy. cInitial IF after pronase digestion showed 1–2+ C3 (n = 2), co-dominant 2+ IgG (n = 2), or bright 3+ IgG (n = 4); re-biopsy in 3 patients showed C3 staining 3–4+ in all with or without absent IgG staining

593Pediatr Nephrol (2021) 36:591–600

These included 18 biopsies from 2007 to 2013 initially diag- nosed as MPGN types I–III that were re-classified. Figure 1 shows flow of patient enrolment and classification. Of 92 pa- tients, 48 (52.2%) were classified as DDD, 26 (28.3%) as C3GN, and 18 (19.6%) as IC-MPGN. One patient with DDD, mimicking necrotizing vasculitis, has been reported previously [18]. Clinical and histological features of patients are shown in Table 1. Patients presented at all ages (range 4.1–18 years); nine patients were < 6 years old. A minor prodromal illness, most frequently fever and upper respiratory tract infection, was noted in 29 (31.5%). Chief presenting features were nephrotic syn- drome (65.2%), acute GN (15.2%), and rapidly progressive GN (15.2%). At presentation, 14 (15.2%) patients required dial- ysis. Most had significant hypertension and eight had hyperten- sive encephalopathy with seizures.

Rapidly progressive GN (with 40–100% crescents on bi- opsy), gross hematuria, and dialysis dependence were signif- icantly higher in patients with DDD compared with C3GN (Table 1). As compared with IC-MPGN, more patients with C3 glomerulopathy (DDD and C3GN) showed low C3 at on- set (< 90 mg/dL; 85.7% versus 41.1%, P < 0.001), which persisted during follow-up (Table 1). No patient had family history of similar illness or any other autoimmune disease.

Biopsy characteristics

Histopathological findings are shown in Table 2. Of 33 biop- sies showing any crescents, 27 (81.8%) were classified as DDD. Classification based on IF and EM is shown in Fig. 1. In patients classified by IF alone, 8 (18.2%) with DDD and

Table 1 Baseline clinical and biochemical parameters in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy (N = 74) IC-MPGN (N = 18) P† P‡

DDD (N = 48) C3GN (N = 26)

Age at onset, yr 9.6 (7.6, 11.4) 10.0 (8.2, 13.1) 9.5 (7.3, 10.3) 0.13 0.28

Age at diagnosis, yr 9.9 (8.3, 11.6) 10.5 (8.7, 13) 9.6 (7.9, 11.0) 0.21 0.39

Boys 24 (50) 19 (73.1) 13 (72.2) 0.05 0.27

Prodromal symptoms 20 (41.7) 5 (19.2) 4 (22.2) 0.05 0.34

First clinical manifestationa 0.05 0.45 Nephrotic syndrome 26 (54.2) 20 (76.9) 14 (77.8)

Rapidly progressive GN 12 (25.0) 1 (3.8) 1 (5.6)

Acute GN 6 (12.5) 5 (19.2) 3 (16.7)

Chronic GN 3 (6.3) – –

Hematuria 0.05 0.37 None 7 (14.6) 4 (15.4) 1 (5.6)

Microscopic 26 (54.2) 20 (76.9) 15 (83.3)

Gross 15 (31.3) 2 (7.7) 2 (11.1)

Hypertension 0.24 0.54 None 9 (18.8) 7 (26.9) 2 (11.1)

Stage 1 16 (33.3) 13 (50.0) 7 (38.9)

Stage 2 23 (47.9) 6 (23.1) 9 (50)

eGFRcr, mL/min/1.73 m2 b 79 (60, 94) 101 (65, 133) 89 (76, 121) 0.06 0.60

eGFRcr < 60 mL/min/1.73 m2 19 (39.6) 6 (23.1) 3 (16.7) 0.15 0.16

Serum albumin, g/dL 1.9 (1.7, 2.6) 2.4 (1.8, 3.0) 2.1 (1.8, 2.5) 0.13 0.66

24-h urine protein, g/dayc 2.2 (1.5, 3.9) 2.0 (1.5, 3.3) 2.4 (1.9, 4.5) 0.68 0.16

Serum C3, mg/dLd 29 (15, 56) 27.5 (17, 61.5) 91 (32.5, 109.5) 0.86 <0.001

Persistently low C3e 25 (53.2) 14 (53.8) 3 (16.7) 0.60 0.001

Numbers are N (%) or median (first, third quartiles)

eGFRcr estimated glomerular filtration rate; GN glomerulonephritis †Comparison between DDD and C3GN ‡Comparison between C3 glomerulopathy and IC-MPGN aOne patient with DDD presented with isolated gross hematuria b Computed in non-dialysis dependent patients c Proteinuria quantified in 33 DDD, 18 C3GN, and 11 IC-MPGN at baseline d Two patients with DDD and one with C3GN had serum C3 levels < 15 mg/dL; not available in 5 patients e Persistently low C3 defined as ≥ 2 low values (< 90 mg/dL) over ≥ 6 months; not available for one patient with DDD

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one (3.8%) with C3GN were misclassified as IC-MPGN due to lower intensity of C3 staining compared with IgG. Four of these 9 patients underwent a re-biopsy, 12–51.4 months later, which showed increased C3 staining to 3–4+ in all and two- order decrease in intensity of IgG staining in three patients (Fig. 1). Therefore, IF alone correctly classified 87.1% pa- tients with DDD (81.8% sensitivity, 100% specificity).

DDD was characterized by dense osmiophilic deposits along the lamina densa that were continuous in 38 or discon- tinuous in 10 patients; additional subepithelial and subendothelial deposits were noted in 7 patients, each. Electron dense deposits in C3GN were mesangial in 20, subendothelial 19, light intramembranous 7, paramesangial 6, and subepithelial in 4 biopsies. Deposits in IC-MPGNwere subendothelial in 10, mesangial 9, subepithelial 3, and paramesangial in 2 biopsies.

A total of 26 patients underwent a second kidney biopsy after median of 36.1 (16.8, 61.2) months. Indications included unsatisfactory EM on initial biopsy in 10 patients (5 DDD, 4 C3GN, and 1 IC-MPGN), for assessing disease progression in

15, and post-transplant recurrence in one. Original pattern on light microscopy was preserved in all; one patient showed recurrence of crescentic pattern of kidney injury. Re-biopsies showed persistence of neutrophilic infiltration and endocapillary proliferation in 12 and 8 patients, respectively. There was an increase in grade of interstitial fibrosis and/or tubular atrophy in 11 biopsies, and median global sclerosis increased from 0 to 10%. The IF pattern was consistent with the initial biopsy in four and seven patients with IC-MPGN and C3GN, respectively, but altered in four patients initially classified as IC-MPGN (described previously).

Therapy

Initial therapy comprisedMMF in 51, tacrolimus in 11, and IV cyclophosphamide in 20 patients with additional IV methyl- prednisolone in 15 and plasma exchanges in 6 patients (Table 3). Induction with IV cyclophosphamide was followed by therapy with MMF in 11, tacrolimus in 2, and azathioprine in one patient. Patients with non-response were switched to

Table 2 Light microscopic findings in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy (N = 74) IC-MPGN (N = 18) P† P‡

DDD (N = 48) C3GN (N = 26)

No. of glomeruli 19 (13.5, 27) 17.5 (13.5, 28.5) 11 (15, 23) 0.47 0.36

Patterna

Membranoproliferative GN 35 (72.9) 23 (88.5) 17 (94.4) 0.12 0.16

Diffuse proliferative GN 5 (10.4) 2 (7.7) 1 (5.6) 0.70 0.63

Crescentic GN 7 (14.6) 1 (3.8) – 0.16 –

Crescents (% glomeruli) <0.001 0.33

None 21 (43.8) 25 (96.2) 13 (72.2)

< 50 20 (41.7) – 5 (27.8)

≥ 50 7 (14.6) 1 (3.8) –

Endocapillary proliferation 27 (56.3) 15 (57.7) 12 (66.7) 0.76 0.60

Neutrophilic infiltration 38 (79.2) 18 (69.2) 8 (44.4) 0.49 0.02

Focal sclerosis 8 (16.7) 5 (19.2) – 0.75 –

Global sclerosis (% glomeruli) 0.36 0.09

None 28 (58.3) 18 (69.2) 7 (38.9)

< 50 17 (35.4) 8 (30.8) 11 (61.1)

≥ 50 3 (6.3) – –

Interstitial fibrosis 0.21 0.54

None 28 (58.3) 19 (73.1) 9 (50.0)

Grade 1 13 (27.1) 7 (26.9) 7 (38.9)

Grade 2 5 (10.4) – 2 (11.1)

Grade 3 2 (4.2) – –

GN glomerulonephritis

Numbers are median (first, third quartiles) or N (%) †Comparison between DDD and C3GN ‡Comparison between C3 glomerulopathy and IC-MPGN a Sclerosed glomeruli in one patient with DDD not allowing interpretation

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therapy with MMF in 2, tacrolimus 26, IV cyclophosphamide 7, rituximab 6, and azathioprine in 3 patients.

In patients with C3 glomerulopathy, initial therapy with MMF and tacrolimus were administered for 19.7 (12.0, 35.9) and 20.3 (9.9, 25.4) months, respectively. Complete or partial remission was achieved in 11/40 (27.5%) patients with MMF and 5/10 (50%) patients with tacrolimus. IV cyclophos- phamide was preferentially used in patients with C3 glomer- ulopathy (n = 16) and induced remission in 6 (37.5%) patients.

Of 11 patients with IC-MPGN initially treated with MMF, 3 (27.3%) achieved complete or partial remission. One of four patients administered IV cyclophosphamide achieved partial remission and one receiving tacrolimus remained non- responsive.

Outcome

Median duration of follow-up was 4.3 (2.3, 7.2) years. Proportion of patients with DDD, C3GN, and IC-MPGN in complete or partial remission at last follow-up were 28.5,

36.1, and 16.7%, respectively [log-rank P = 0.54, Fig. 2(a)]. Parameters associated with complete or partial remission at last follow-up are shown in Table 4. On multivariable analy- sis, albumin < 2.5 g/dL at onset (HR 0.29; P = 0.005) and persistently low serum C3 (HR 0.34; P = 0.02) were indepen- dently associated with non-response. Of 9 patients (5 DDD, 3 C3GN, and 1 IC-MPGN) who were lost to follow-up after median of 2.3 (2.0, 2.5) years, 5 had achieved complete or partial remission. Proteinuria recurred following remission in 15 patients after median of 23.8 (13.2–42.5) months; 13 remained non-responsive.

Median kidney survival in patients with DDD was 6.9 years. The 5-year kidney survival was 62.6, 85.5, and 88.5% in patients with DDD, C3GN, and IC-MPGN, respec- tively [log-rank P = 0.006, Fig. 2(b)]. Five patients (3 DDD, 2 IC-MPGN), all with CKD stage 5, died after median of 3.5 (3.4, 6.1) years due to sepsis or complications of kidney fail- ure. On multivariable analysis, presentation as rapidly pro- gressive GN (HR = 11.2; P < 0.001), age > 10 years at onset (HR = 4.0; P = 0.004), and DDD (HR = 4.2; P = 0.02) were independently associated with adverse outcome (Table 4);

Table 3 Therapy and outcome in patients with dense deposit disease (DDD), C3 glomerulonephritis (C3GN), and immune-complex membranoproliferative glomeru- lonephritis (IC-MPGN)

C3 glomerulopathy IC-MPGN P

DDD C3GN

Therapya N = 48 N = 26 N = 18

Mycophenolate mofetil 22 (45.8) 18 (69.2) 11 (61.1)

Tacrolimus 4 (8.3) 6 (23.1) 1 (5.6)

IV cyclophosphamideb +maintenance immunosuppressionc 14 (29.2) 2 (7.7) 4 (22.2)

Prednisolone alone 4 (8.3) – 2 (11.1)

6 months follow-up N = 48 N = 26 N = 18 0.95

Complete remission 6 (12.5) 3 (11.5) 1 (5.6)

Partial remission 8 (16.7) 4 (15.4) 3 (16.7)

Non-response 34 (70.8) 17 (73.1) 14 (77.8)

eGFRcr < 15 mL/min/1.73 m2 or death 6 (12.5) 1 (3.9) – 0.16

12-months follow-up N = 48 N = 25 N = 17 0.49

Complete remission 8 (16.7) 2 (8.0) 2 (11.8)

Partial remission 10 (20.8) 5 (20.0) 1 (5.9)

Non-response 30 (62.5) 18 (72.0) 14 (82.3)

eGFRcr < 15 mL/min/1.73 m2 or death 7 (14.6) 1 (4.0) – 0.12

Last follow-up N = 48 N = 26 N = 18 0.78

Complete remission 5 (10.4) 3 (11.5) 2 (11.1)

Partial remission 8 (16.7) 5 (19.2) 1 (5.6)

Non-response 35 (72.9) 18 (69.2) 15 (83.3)

eGFRcr < 15 mL/min/1.73 m2 or death 19 (39.6) 2 (7.7) 2 (11.1) 0.003

Numbers are N (%) or median (first, third quartiles)

eGFRcr, estimated glomerular filtration rate a Other therapies were IV rituximab (n = 1, DDD) or no immunosuppression (n = 2 DDD, n = 1 C3GN) bAdditional 3–6 doses of IV methylprednisolone (n = 15) or plasma exchanges (n = 6) cMaintenance immunosuppression, begun if eGFRcr > 30 mL/min/1.73 m2 at 6 months, comprised mycopheno- late mofetil (n = 11), tacrolimus (n = 2), azathioprine (n = 1), and prednisolone (n = 1)

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achieving complete or partial remission was protective (HR = 0.04; P < 0.001).

Discussion

We report a single center experience of 92 children with C3 glomerulopathy and IC-MPGN. Patients with DDD com- prised almost half of the cohort and were more likely to

present with rapidly progressive GN. Immunofluorescence misclassified 18.2% patients with DDD as IC-MPGN, highlighting the histological overlap between C3 glomerulop- athy and IC-MPGN. One-fourth of the patients achieved com- plete or partial remission following immunosuppressive ther- apy; rate of remission was similar in patients with C3 glomer- ulopathy and IC-MPGN. Rapidly progressive GN, older age (> 10 years), DDD, and lack of remission were independently associated with progression to CKD stage 5 or death.

a

b

Fig. 2 Probability of complete or partial remission at last follow-up and kidney survival free of ad- verse outcomes. (a) Complete or partial remission at 6 months, 12 months, and last follow-up was achieved in 18.9, 23.3, and 28.5% patients with DDD, re- spectively (continuous line). Corresponding proportions in pa- tients with C3GN and IC-MPGN were 15.4, 19.6, and 36.1% and 11.1, 16.7, and 16.7%, respec- tively (interrupted lines, log-rank P = 0.54). (b) The 1- and 5-year kidney survival was 81.3 and 62.6% in patients with DDD (continuous line), 96.2 and 85.5% in C3GN, and 100 and 88.5% in IC-MPGN (interrupted lines), re- spectively (log-rank P = 0.006). Kidney survival in patients with C3GN and IC-MPGNwas similar (log-rank, P = 0.93)

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Clinical features of patients in the current study showed similarities with previously reported pediatric and adult series. Nephrotic syndrome was the chief presentation in 65.2% of our patients similar to other pediatric reports (22–69.7%) [19–21]. We observed similar frequency of nephrotic syn- drome in C3 glomerulopathy (62%) and IC-MPGN (78%). In mixed pediatric and adult cohorts, nephrotic syndrome was more frequent in IC-MPGN (43–70%) compared with C3 glomerulopathy (26–52%) [1, 9, 22]. One-fourth of pa- tients with DDD presented with rapidly progressive GN, and 14.6% showed more than 50% crescents. The prevalence of crescentic GN in DDD is reported as 6–21% in adults and children [11, 19, 21, 23]. Majority had hematuria (86.9%) and significant hypertension (41.3%), comparable to 38– 100% and 55–79%, respectively, in other pediatric series [7, 19, 21, 23]. Acute GN, variably reported as 17–61% at onset [20, 21, 23], occurred in 15% of patients in this study. Levels of serum C3 were lower in patients with C3 glomerulopathy compared with IC-MPGN but similar in DDD and C3GN. Other reports suggest that C3 levels in DDD are lower than C3GN [9, 11]. About 40% of patients with IC-MPGN had low C3 at onset, similar to previous studies [9, 24, 25].

An infectious trigger at onset occurred in 31.5% of the present patients, similar to 28–57% in pediatric cohorts from

Europe and North America [7, 20, 21]. It is suggested that an infectious trigger might lead to sustained activation of the alternative pathway and result in persistent glomerular inflam- mation even after the infection is controlled. Infectious trig- gers activate the classical or lectin pathways which may ex- plain the bright IgG staining in comparison to C3 deposits on eight biopsies of patients with DDD. In support of this hy- pothesis, we previously reported bright glomerular C4d stain- ing in biopsies with C3 glomerulopathy [18]. Thus, the IF criteria of C3 dominance at least two orders of magnitude stronger than other immune deposits had a sensitivity of 81.8% for DDD in the present cohort, similar to 88% reported by Hou et al. [3]. In addition, as reported previously, three patients with C3 glomerulopathy had transient IgG staining at onset that disappeared on re-biopsy [24]. Due to the evolu- tion of IF findings over time, the consensus guidelines on C3 glomerulopathy suggest using EM to distinguish DDD from C3GN [13].

The treatment of these conditions is challenging. While MMF was shown to induce remission in 45–86% adults with C3 glomerulopathy in retrospective series [10, 26, 27], its efficacy was not replicated in another cohort where 25% of patients with more severe disease progressed to kidney failure [28]. MMF was the initial therapy in 62% of the present

Table 4 Association of remission and progression to chronic kidney disease (CKD) stage 5 or death

Parameter Complete or partial remission at last follow-up CKD stage 5 or death

Univariate analysis Multivariable analysis Univariate analysis

Multivariable analysis

Baseline Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P Hazards ratio (95% CI)

P

Age > 10 years 1.27 (0.57, 2.83) 0.56 3.77 (1.57, 9.03) 0.003 3.98 (1.55, 10.25) 0.004 Gross hematuria 2.26 (0.63, 8.16) 0.21 1.25 (0.49, 3.18) 0.65 Onset serum albumin < 2.5 g/dL 0.40 (0.18, 0.89) 0.03 0.29 (0.12, 0.69) 0.005 0.63 (0.33, 1.25) 0.19 24-h urine protein at onset 1.00 (0.99, 1.00) 0.58 1.00 (0.99, 1.00) 0.85 Low C3 at onset (< 90 mg/dL) 0.66 (0.28, 1.60) 0.36 1.00 (0.39, 2.56) 0.99 Rapidly progressive GNa 1.83 (0.68, 4.92) 0.23 3.23 (1.26, 8.29) 0.02 11.19 (3.21, 39.01) < 0.001 Onset eGFRcr < 30 mL/min/1.73 m2 1.30 (0.44, 3.81) 0.64 6.14 (2.40, 15.74) < 0.001 1.76 (0.48, 6.53) 0.40b

Histopathology Dense deposit diseasec 1.84 (0.52, 6.46) 0.34 4.67 (1.09, 20.1) 0.04 4.21 (1.29, 13.69) 0.02 C3 glomerulonephritisc 2.05 (0.54, 7.74) 0.29 0.93 (0.13, 6.64) 0.94 Neutrophilic infiltration 1.65 (0.62, 4.42) 0.32 1.43 (0.53, 3.89) 0.48 Endocapillary proliferation 0.81 (0.36, 1.81) 0.61 1.19 (0.51, 2.78) 0.68 Interstitial fibrosis, tubular atrophy stages 2–3

0.67 (0.27, 1.69) 0.40 5.83 (2.37, 14.39) < 0.001 2.76 (0.99, 7.64) 0.05

Therapy and follow-upd

Persistently low C3 0.36 (0.15, 0.90) 0.03 0.34 (0.13, 0.84) 0.02 1.43 (0.53, 3.89) 0.48 Therapy with MMF 0.66 (0.30, 1.47) 0.31 0.51 (0.22, 1.19) 0.12 Therapy with tacrolimus 1.49 (0.51, 4.37) 0.47 0.55 (0.23, 1.32) 0.18 Complete or partial remission 0.12 (0.03, 0.49) 0.004 0.04 (0.01, 0.23) < 0.001

eGFRcr, estimated glomerular filtration rate; GN, glomerulonephritis; MMF, mycophenolate mofetil a All patients had > 40% crescents b eGFRcr < 30 mL/min/1.73 m2 was not significant in the multivariable model and therefore excluded c Compared with IC-MPGN as reference d Therapy with IV cyclophosphamide was not considered due to preferential use in patients with rapidly progressive GN

598 Pediatr Nephrol (2021) 36:591–600

patients among whom 27.5% attained complete or partial re- mission; response rates in C3 glomerulopathy and IC-MPGN were similar. Therapy with tacrolimus showed remission in 5 of 11 (45.5%) patients; variable remission rates of 11–94% are reported in adults [12, 26, 29]. Cyclophosphamide has shown conflicting results in adults with up to 78% complete remis- sion [30], but without improvement in kidney survival [31]. We used IV cyclophosphamide, chiefly in patients with severe disease, which induced complete or partial remission in 35% of patients. While complement blockade with eculizumab ap- pears to be promising in patients with severe disease, especial- ly crescentic GN [14], this is not available in our country.

Complete or partial remission occurred in 28.5% patients with DDD, 36.1% in C3GN, and 16.7% in IC-MPGN at last follow-up in the present cohort. In other pediatric studies, rate of remission following immunosuppressive therapy was re- ported as 22–78% [7, 19–21, 23, 32]. In a multivariable Cox proportional hazard model, we showed that serum albumin < 2.5 g/dL at onset and persistently low levels of C3 were inde- pendent factors associated with non-response to therapy; such patients may require aggressive management. In addition to the histological overlap, we showed that kidney survival of patients with IC-MPGN and C3GN was similar. In contrast, 67–88% of patients with C3GN in Japanese pediatric cohorts progressed to CKD stage 5 as compared to 32–50% of patients with IC-MPGN [24, 25]. While DDD was associated with a poor kidney survival in the present study, similar to previous observations [11, 33], others show no difference in survival between C3GN and DDD [4, 9, 10, 21]. Progressive kidney failure occurred in 29–50% patients by 2–11 years in other cohorts [9–12, 34]. Similar to previous studies, we observed rapidly progressive GN, older age, and non-response to ther- apy were other independent factors associated with progres- sion to CKD stage 5 or death [9–11, 19, 21, 28].

This study was limited by its retrospective and single- center design. While EM-based classification was done in all 92 patients, biopsies that were inadequate for EM were not included and may have led to selection bias. Approximately 40% of patients were enrolled in the last four years and 9.8% were lost to follow-up; therefore, the median duration of follow-up was rather short (4.3 years). We did not perform assays for C3Nef, sC5b-9, autoantibodies to factor B, and genetic testing for complement regulatory genes or copy num- ber variations in CFHR1–5. Clinical utility of results of such testing in making therapeutic decisions is as yet unclear.

In summary, we present clinicopathological phenotype and outcome of children with C3 glomerulopathy and IC-MPGN. We found that immunofluorescence misclassified one fifth of patients with DDD as IC-MPGN, highlighting the histological overlap of C3 glomerulopathy and IC-MPGN. While DDD was independently associated with progressive kidney failure, outcomes were similar in IC-MPGN and C3GN. Rapidly pro- gressive GN, older age, and non-response to therapy were

additionally associated with adverse outcome. Response to immunosuppression was unsatisfactory irrespective of histo- logical classification or the type of immunosuppressive thera- py, therefore, emphasizing the need for disease-specific ther- apies, complement inhibitors or modulators.

Acknowledgments The study received funding support from the Department of Biotechnology, Government of India [BT/PR11030/ MED/30/1644/2016].

Compliance with ethical standards The study was conduct- ed following the Institute ethical clearance (IEC/NP-353/08-10-2014).

Conflict of interest The authors declare that they have no conflict of interest.

References

1. Fakhouri F, Le Quintrec M, Fremeaux-Bacchi V (2020) Practical management of C3 glomerulopathy and immunoglobulin-mediated MPGN: facts and uncertainties. Kidney Int. https://doi.org/10.1016/ j.kint.2020.05.053

2. Smith RJH, Appel GB, Blom AM, Cook HT, D'Agati VD, Fakhouri F, Fremeaux-Bacchi V, Jozsi M, Kavanagh D, Lambris JD, Noris M, Pickering MC, Remuzzi G, de Cordoba SR, Sethi S, Van der Vlag J, Zipfel PF, Nester CM (2019) C3 glomerulopathy— understanding a rare complement-driven renal disease. Nat Rev Nephrol 15:129–143

3. Hou J, Markowitz GS, Bomback AS, Appel GB, Herlitz LC, Barry Stokes M, D'Agati VD (2014) Toward a working definition of C3 glomerulopathy by immunofluorescence. Kidney Int 85:450–456

4. Bomback AS, Appel GB (2012) Pathogenesis of the C3 glomeru- lopathies and reclassification of MPGN. Nat Rev Nephrol 8:634– 642

5. Pickering MC, D'Agati VD, Nester CM, Smith RJ, Haas M, Appel GB, Alpers CE, Bajema IM, Bedrosian C, Braun M, Doyle M, Fakhouri F, Fervenza FC, Fogo AB, Fremeaux-Bacchi V, Gale DP, Goicoechea de Jorge E, Griffin G, Harris CL, Holers VM, Johnson S, Lavin PJ, Medjeral-Thomas N, Paul Morgan B, Nast CC, Noel LH, Peters DK, Rodriguez de Cordoba S, Servais A, Sethi S, Song WC, Tamburini P, Thurman JM, Zavros M, Cook HT (2013) C3 glomerulopathy: consensus report. Kidney Int 84: 1079–1089

6. Sethi S, Fervenza FC (2011) Membranoproliferative glomerulone- phritis: pathogenetic heterogeneity and proposal for a new classifi- cation. Semin Nephrol 31:341–348

7. Holle J, Berenberg-Gossler L, Wu K, Beringer O, Kropp F, Muller D, Thumfart J (2018) Outcome of membranoproliferative glomer- ulonephritis and C3-glomerulopathy in children and adolescents. Pediatr Nephrol 33:2289–2298

8. Iatropoulos P, Noris M,Mele C, Piras R, Valoti E, Bresin E, Curreri M, Mondo E, Zito A, Gamba S, Bettoni S, Murer L, Fremeaux- Bacchi V, Vivarelli M, Emma F, Daina E, Remuzzi G (2016) Complement gene variants determine the risk of immunoglobulin- associated MPGN and C3 glomerulopathy and predict long-term renal outcome. Mol Immunol 71:131–142

9. Servais A, Noel LH, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey MA, Macher MA, Zuber J, Karras A, Provot F, Moulin B, Grunfeld JP, Niaudet P, Lesavre P, Fremeaux-Bacchi V (2012) Acquired and genetic complement abnormalities play a

599Pediatr Nephrol (2021) 36:591–600

critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82:454–464

10. Bomback AS, Santoriello D, Avasare RS, Regunathan-Shenk R, Canetta PA, Ahn W, Radhakrishnan J, Marasa M, Rosenstiel PE, Herlitz LC, Markowitz GS, D'Agati VD, Appel GB (2018) C3 glomerulonephritis and dense deposit disease share a similar dis- ease course in a large United States cohort of patients with C3 glomerulopathy. Kidney Int 93:977–985

11. Medjeral-Thomas NR, O'Shaughnessy MM, O'Regan JA, Traynor C, Flanagan M, Wong L, Teoh CW, Awan A, Waldron M, Cairns T, O'Kelly P, Dorman AM, Pickering MC, Conlon PJ, Cook HT (2014) C3 glomerulopathy: clinicopathologic features and predic- tors of outcome. Clin J Am Soc Nephrol 9:46–53

12. Ravindran A, Fervenza FC, Smith RJH, De Vriese AS, Sethi S (2018) C3 glomerulopathy: ten years' experience at Mayo Clinic. Mayo Clin Proc 93:991–1008

13. Goodship TH, Cook HT, Fakhouri F, Fervenza FC, Fremeaux- Bacchi V, Kavanagh D, Nester CM, Noris M, Pickering MC, Rodriguez de Cordoba S, Roumenina LT, Sethi S, Smith RJ, Conference Participants (2017) Atypical hemolytic uremic syn- drome and C3 glomerulopathy: conclusions from a “Kidney dis- ease: improving global outcomes” (KDIGO) controversies confer- ence. Kidney Int 91:539–551

14. Le Quintrec M, Lapeyraque AL, Lionet A, Sellier-Leclerc AL, Delmas Y, Baudouin V, Daugas E, Decramer S, Tricot L, Cailliez M, Dubot P, Servais A, Mourey-Epron C, Pourcine F, Loirat C, Fremeaux-Bacchi V, Fakhouri F (2018) Patterns of clinical re- sponse to eculizumab in patients with C3 glomerulopathy. Am J Kidney Dis 72:84–92

15. Habib R, Kleinknecht C, Gubler MC, Levy M (1973) Idiopathic membranoproliferative glomerulonephritis in children. Report of 105 cases. Clin Nephrol 1:194–214

16. Flynn JT, Kaelber DC, Baker-Smith CM, Blowey D, Carroll AE, Daniels SR, de Ferranti SD, Dionne JM, Falkner B, Flinn SK, Gidding SS, Goodwin C, Leu MG, Powers ME, Rea C, Samuels J, Simasek M, Thaker VV, Urbina EM, Subcommittee on Screening and Management of High Blood Pressure in Children (2017) Clinical practice guideline for screening and management of high blood pressure in children and adolescents. Pediatrics 140: e20171904

17. Schwartz GJ, Munoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL (2009) New equations to estimate GFR in children with CKD. J Am Soc Nephrol 20:629–637

18. Singh G, Singh SK, Nalwa A, Singh L, Pradeep I, Barwad A, Sinha A, Hari P, Bagga A, Bagchi S, Agarwal SK, Dinda AK (2019) Glomerular C4d staining does not exclude a C3 glomerulopathy. Kidney Int Rep 4:698–709

19. Caltik Yilmaz A, Aydog O, Akyuz SG, Bulbul M, Demircin G, Oner A (2014) The relation between treatment and prognosis of childhood membranoproliferative glomerulonephritis. Ren Fail 36:1221–1225

20. Nicolas C, Vuiblet V, Baudouin V, Macher MA, Vrillon I, Biebuyck- GougeN,DehennaultM,Gie S,MorinD,NivetH,Nobili F,Ulinski T, Ranchin B, Marinozzi MC, Ngo S, Fremeaux-Bacchi V, Pietrement C (2014) C3 nephritic factor associated with C3 glomerulopathy in chil- dren. Pediatr Nephrol 29:85–94

21. Nasr SH, Valeri AM, Appel GB, Sherwinter J, Stokes MB, Said SM, Markowitz GS, D'Agati VD (2009) Dense deposit disease: clinicopathologic study of 32 pediatric and adult patients. Clin J Am Soc Nephrol 4:22–32

22. Iatropoulos P, Daina E, Curreri M, Piras R, Valoti E, Mele C, Bresin E, Gamba S, Alberti M, Breno M, Perna A, Bettoni S, Sabadini E, Murer L, Vivarelli M, Noris M, Remuzzi G, Registry of Membranoproliferative Glomerulonephritis CG, Nastasi (2018) Cluster analysis identifies distinct pathogenetic patterns in C3 g l o m e r u l o p a t h i e s / i m m u n e c o m p l e x - m e d i a t e d membranoproliferative GN. J Am Soc Nephrol 29:283–294

23. Yazilitas F, Kargin Cakici E, Kurt Sukur ED, Can G, Gungor T, Orhan D, Bulbul M (2020) C3 glomerulopathy: experience of a pediatric nephrology center. Acta Clin Belg. https://doi.org/10. 1080/17843286.2020.1713450

24. Kawasaki Y, Kanno S, OnoA, Suzuki Y, Ohara S, SatoM, Suyama K, Hashimoto K, HosoyaM (2016) Differences in clinical findings, pathology, and outcomes between C3 glomerulonephritis and membranoproliferative glomerulonephritis. Pediatr Nephrol 31: 1091–1099

25. Okuda Y, Ishikura K, Hamada R, Harada R, Sakai T, Hamasaki Y, Hataya H, Fukuzawa R, Ogata K, Honda M (2015) Membranoproliferative glomerulonephritis and C3 glomerulone- phritis: frequency, clinical features, and outcome in children. Nephrology (Carlton) 20:286–292

26. Avasare RS, Canetta PA, Bomback AS, Marasa M, Caliskan Y, Ozluk Y, Li Y, Gharavi AG, Appel GB (2018) Mycophenolate mofetil in combination with steroids for treatment of C3 glomeru- lopathy: a case series. Clin J Am Soc Nephrol 13:406–413

27. Rabasco C, Cavero T, Roman E, Rojas-Rivera J, Olea T, Espinosa M, Cabello V, Fernandez-Juarez G, Gonzalez F, Avila A, Baltar JM, Diaz M, Alegre R, Elias S, Anton M, Frutos MA, Pobes A, BlascoM,Martin F, Bernis C,MaciasM, Barroso S, de Lorenzo A, Ariceta G, Lopez-Mendoza M, Rivas B, Lopez-Revuelta K, Campistol JM, Mendizabal S, de Cordoba SR, Praga M, Spanish Group for the Study of Glomerular Diseases (GLOSEN) (2015) Effectiveness of mycophenolate mofetil in C3 glomerulonephritis. Kidney Int 88:1153–1160

28. Caliskan Y, Torun ES, Tiryaki TO, Oruc A, Ozluk Y, Akgul SU, Temurhan S, Oztop N, Kilicaslan I, Sever MS (2017) Immunosuppressive treatment in C3 glomerulopathy: is it really effective? Am J Nephrol 46:96–107

29. Bagheri N, Nemati E, Rahbar K, Nobakht A, Einollahi B, Taheri S (2008) Cyclosporine in the treatment of membranoproliferative glo- merulonephritis. Arch Iran Med 11:26–29

30. Faedda R, Satta A, Tanda F, Pirisi M, Bartoli E (1994) Immunosuppressive treatment of membranoproliferative glomeru- lonephritis. Nephron 67:59–65

31. Cattran DC, Cardella CJ, Roscoe JM, Charron RC, Rance PC, Ritchie SM, Corey PN (1985) Results of a controlled drug trial in membranoproliferative glomerulonephritis. Kidney Int 27:436–441

32. Kojc N, Bahovec A, Levart TK (2019) C3 glomerulopathy in chil- dren: is there still a place for anti-cellular immunosuppression? Nephrology (Carlton) 24:188–194

33. Viswanathan GK, Nada R, Kumar A, Ramachandran R, Rayat CS, Jha V, Sakhuja V, Joshi K (2015) Clinico-pathologic spectrum of C3 glomerulopathy—an Indian experience. Diagn Pathol 10:6

34. Tarshish P, Bernstein J, Tobin JN, Edelmann CM Jr (1992) Treatment of mesangiocapillary glomerulonephritis with alternate-day prednisone—a report of the International Study of Kidney Disease in Children. Pediatr Nephrol 6:123–130

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