Clinico-pathological Profile and Outcome of C-3 Glomerulopathy in Indian Children

Introduction

Isolated or dominant C3 deposits on immunofluorescent (IF) microscopy is the phenotype which identifies C3 glomerulopathy (C3GP) that occurs due to dysfunction of the alternate pathway of complement. Evaluation by serology and genetic testing can identify the etiopathogenesis of the alternate pathway dysfunction in many but not in all cases. Complement dysfunction can result from genetic defects leading to deficiencies of complement factors or complement regulatory proteins with low levels of C3, Factor B, Factor H, Factor I, or Membrane cofactor protein. It could also occur due to acquired antibodies to Factor H, Factor B, or C3 convertase (C3 nephritic factor). As facilities to investigate the complement factors are not easily available to clinicians, most cases of C3GN remain etiologically undefined. C3GN is currently broadly classified based on the pattern of electron dense deposits on electron microscopy as DDD with ribbon like intramembranous osmiophilic deposits or as C3GN with variable deposits other than dense intramembranous deposits.[12] Since its formal nomenclature in 2012[1] several case series have been published, largely of adults and adolescent children including two publications from India.[34] Ours is the first Indian pediatric case series describing the clinico-pathological profile and outcome of C3GN in 18 prepubertal children biopsied over a 10-year period.

Methodology

This is a retrospective case series of all children with C3GN seen over a period of 10 years in the Division of Pediatric Nephrology at a tertiary care children's hospital. All renal biopsies done at our Centre from January 1, 2007 to December 31, 2016 were reviewed. The immunofluorescent microscopy (IF) results were noted for the presence or absence of C3, IgG, IgM, IgA, and C1q. The intensity of the immunoreactants was graded on a scale of 0, trace, 1+, 2+, 3+.

Results

There were 298 biopsies done during the study period. Twenty-one biopsies were excluded from analysis as the core for IF showed no glomeruli. Of the remaining 277 biopsies, 41 had isolated or dominant C3 deposits on IF. 23/41 were excluded as they were classical postinfectious GN with complete recovery over 12 weeks [Figure 1].

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Figure 1

Flow chart depicting the methodology of selecting children for the study. IF, Immunofluorescence; PIGN, Post infectious glomerulonephritis

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18 biopsies fulfilled the criteria for C3GN. This included 4 with DDD and 14 with C3GN. Seven biopsies were reclassified as C3GN. These included cases that were earlier classified as membranoproliferative glomerulonephritis (MPGN) in 4, mesangioproliferative glomerulonephritis (MesPGN) in 2 and crescentic glomerulonephritis (CGN) in 1 patient.

Kidney biopsy findings

LM showed MesPGN in 7, MPGN in 5, diffuse endocapillary proliferative glomerulonephritis (DPGN) in 3, and CGN in 3. Glomerulosclerosis was seen in 8 (44%), Interstitial Fibrosis and Tubular Atrophy (IFTA) in 5 (27.7%) and both glomerulosclerosis and IFTA in 5 (27.7%).

Isolated C3 deposits were seen on IF in 12 of which 4 had 3+ intensity of deposits. Six patients had dominant C3 (2+/3+) along with immunoglobulins [Figure 2a and b]. EM (done in 14 patients) showed subendothelial EDD in 10, subepithelial in 7, mesangial in 7 and intramembranous in 7 [Figure 2c-e]. Majority of patients had EDD in more than one location [Table 2].

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Figure 2

(a) Immunoflorescence for C3 staining showing 3+ stain in the glomeruli. Original Image, Courtesy: Surgical Pathology Department, Jaslok Hospital. (b) Immunoflorescence for IgG showing 1+ staining in the glomeruli. Original Image, Courtesy: Surgical Pathology Department, Jaslok Hospital. (c) EM image showing Dense deposit disease with ribbon like intramemberanous deposits. Original Image, Courtesy: Electron Microscopy Department, Jaslok Hospital. (d) EM image of a patient with C3 glomerulonephritis, non-DDD with electron dense deposits. Original Image, Courtesy: Electron Microscopy Department, Jaslok Hospital. (e) EM image of patient with C-3 glomerulonephritis with electron dense deposits. Original Image, Courtesy: Electron Microscopy Department, Jaslok Hospital.

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Table 2 Histopathological renal biopsy findings of the patients with C-3 Glomerulopathy

Category	Dense Deposit Disease (n=4)	C3-Glomerulo nephritis (n=14)	Total (n=18)	P
Light Microscopic Finding
Pattern of injury (n, %)
Mesangial Proliferative GN	3 (75.0)	4 (28.6)	7 (38.8)	0.43
Diffuse endocapillary proliferative GN	0 (0)	3 (21.4)	3 (16.7)	0.54
Crescentic GN	0 (0)	3 (21.4)	3 (16.7)	0.54
Membranoproliferative GN	1 (25)	4 (28.6)	5 (27.7)	0.89
Exudative Glomerulonephritis (n, %)	1 (25.0)	11 (78.5)	12 (66.7)	0.42
Mesangial hypercellularity (n, %)	4 (100.0)	11 (78.5)	15 (83.3)	0.32
Glomerulosclerosis (n, %)	4 (100.0)	4 (28.6)	8 (44.4)	0.01
Interstitial Fibrosis, Tubular Atrophy (n, %)	1 (25.0)	4 (28.6)	5 (27.7)	0.89
Immunoflourescence (n, %)
Isolated C3	2 (50.0)	10 (71.4)	12 (66.7)	0.43
C3 + Ig	2 (50.0)	4 (28.6)	6 (35.7)	0.43
Electron Microscopic Finding (n, %)
Mesangial deposits	0 (0)	7 (50.0)	7 (38.9)	0.53
Subepithelial deposits/humps	1 (25.0)	6 (42.9)	7 (38.9)	0.53
Subendothelial deposits	2 (50.0)	8 (57.1)	10 (55.6)	0.81
Intramembranous deposits, DDD	4 (100.0)	3 (21.4)	7 (38.9)	0.006

Renal outcome

Duration of follow up ranged from 2 to 138 months with a median of 38.2 months. Kaplan Meir survival analyses showed that the 1-year patient survival was 88.5%, 5 and 10 years survival was 70.8% respectively. The renal survival without progression to ESRD was 60.2% at 1 year, 48.2% at 5 and 10 years, respectively. (HR 0.38,95% C.I.: 0.1–1.4, P = 0.1) [Figure 3] The survival curves between DDD and C3GN were not statistically different ((HR = 0.44, 95%CI = 0.09-2.23, P = 0.43).

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Figure 3

Comparing the absolute patient survival and renal survival of patients without progression to ESRD in the C-3 Glomerulopathy cohort. The upper curve shows absolute survival of patients, with 1-year survival @ 88.5% and the 5-and 10-year survival @ 70.8%. The lower curve is to represent the renal survival without progression to ESRD, with 1-year, 5-year survival being 60.2% and 48.1%, respectively. (P value = 0.1; HR 0.38, C.I.: 0.1-1.4)

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The renal survival at 5 years in children with initial presentation with hematuria was 80% as compared to those with initial presentation as nephrotic (57%), nephritic (0%) or AKI (33.3%) (P = 0.4) [Figure 4]. Using the Cox regression hazard model, the only independent variable that was identified to adversely affect the renal survival was IFTA on biopsy (P = 0.013, HR 8.1; 95%CI 1.6-42).

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Figure 4

Comparing the renal survival of C-3 Glomerulopathy patients based on their different symptoms at presentation. The curves above show no statistically significant difference (P value = 0.4) in the renal survival. The renal survival without progression to ESRD in hematuria patients was 80%, nephrotic 57%, AKI 33% and nephritic 0%

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Discussion

In the last 15 years, there have been several large published case series of adults with C3GN.[7891011] Pediatric case series have been small and have not included many prepubertal children. Differences have been noted in the clinicopathological phenotype between adults and children. Long-term outcome is variable in both, with a high rate of progression to ESRD over 10 years.

Although C3GN is an uncommon entity, its prevalence appears to be greater in children than in adults. C3GN accounted for 5.3% of all renal biopsies in our study and 4.6% in another pediatric series.[12] This is higher than the prevalence of around 1% in most adult studies.[34810] We do not know if this indicates an increased prevalence of C3GN in children or is merely a reflection of more restrictive indications for biopsy in children when compared to adults.

We found that the triad of hematuria, proteinuria and low C3 was a sensitive marker for the presence of C3GN. It was seen in 88% of all our patients and in 100% of patients with DDD. The common finding of hematuria, proteinuria and low C3 has also been noted in other pediatric studies.[78] C3 was normal in two of our patients. The presence of C3GN in the absence of low serum C3 is now a known entity in certain genetic variants such as CHFR gene mutations.[813] Low C3 is less common in adults and was found in only sixty six percent of the patients in a large series.[8]

As noted in most studies, C3GN was the dominant subtype seen in 77.8% of our patients.[8111415] DDD was seen in twenty-two percent of our cohort. DDD has been reported as an ultra-rare disease that is slightly more common in children and in young adults. Hematuria was seen in all cases of DDD. Although non-nephrotic proteinuria was seen at presentation in half the cases, the proteinuria increased to nephrotic range at follow up.

MPGN is often considered as the typical light microscopic finding in C3GN. However, it is not always the commonest light microscopic change seen in all C3GN studies, especially in children.[1617] Walker et al. in a review of 80 cases of DDD (74% children) from different parts of the world concluded that DDD is not synonymous with MPGN and that MesPGN was the commonest LM finding seen in 45% of cases.[17] In the study by Nasr et al., MPGN was the commonest pathology in adults but MesPGN was seen as commonly as MPGN in children.[7] The most common morphology on LM in our study was MesPGN, seen in 75% of our children with DDD.

The prevalence of chronicity changes in our study was much higher than what is reported in other series suggesting delayed referral and late recognition of the disease.[7] In a Korean study of nine children with DDD, no patient had evidence of chronicity on histopathology and none progressed to ESRD. This may have been due to very early detection as these children were diagnosed in a school screening program.[18]

Forty percent of our children with C3GN had a steep progression to ESRD in the first year after diagnosis. Steep progression was noted by Lu et al. in DDD with 25% reaching ESRD by 2.5 years and 33% by 5 years.[9] In our patients the rapid decline was commoner in C3GN than in DDD probably related to the occurrence of crescentic GN in this group. Children with crescentic GN and AKI at onset failed to recover despite pulse steroids, cyclophosphamide with plasma exchange, and progressed to ESRD. Published studies on the results of plasma infusions for factor deficiencies and plasma exchange with immunosuppression for antibody mediated C3GN have shown benefit in some but not in all patients.[1920] Anticomplement therapy with Eculizumab, a C5a inhibitor, to prevent formation of membrane attack complexes seems an attractive option for the disease that is mediated by complement pathway dysfunction. Dramatic improvement has been noted in many, but not all patients have benefitted.[122122] The impact of Eculizumab in more chronic cases is not uniformly favorable.

Currently, there are no evidence-based recommendations for the management of this rare disease. McEnery and Adams had shown the beneficial effect of long-term alternate day prednisolone on six children with DDD who achieved complete remission.[23] Remission was also reported in 46.9% of children in Nasr's cases of DDD.[7] Two patients with DDD in our study achieved complete remission with long-term alternate day steroids. However, not all our patients had a good outcome with steroids alone.

The best response in our study was seen in five children who received long-term mycophenolate mofetil along with alternate day steroids. All five achieved remission (complete in 3 and partial in 2) with normalization of C3 levels. A Spanish study showed beneficial effects in adults treated with mycophenolate mofetil when compared to other immunosuppressive agents or no immunosuppression (80% vs 50% vs 20%).[24] Not all studies have shown good results and evidence of benefit from the use of antiproliferative drugs such as mycophenolate is conflicting.[12162425] KDIGO recommends that steroids and mycophenolate mofetil could be tried in cases with moderate severity.[16]

The overall 5-year renal survival of 52% in our study was lower than the renal survival reported in many other studies.[7814] The 5-year renal survival was better in DDD (75%) compared to those with C3GN (40%). This is at variance with most other studies which have shown that prognosis is worse in DDD when compared to C3GN. In some studies children with DDD seem to have fared better than the adults.[789] Despite the apparent better renal survival in the medium term, the long-term prognosis in our patients with DDD remains guarded as morphological changes of chronicity such as glomerulosclerosis was seen in all DDD patients. However, the only predictor of adverse outcome in our study was the presence of IFTA on renal biopsy. Adverse impact of low eGFR at presentation and IFTA on biopsy have also been reported in other studies.[11]

In conclusion, C3 glomerulopathy in children is a serious disease with significant risk of progression to ESRD. The beneficial role of early introduction of long-term steroids along with mycophenolate needs to be assessed in prospective studies.