Background: Membranoproliferative glomerulonephritis (MPGN) with immune complexes and C3 glomerulopathy (C3G) in children are rare and have a variable outcome, with some patients progressing to end-stage renal disease (ESRD). Mutations in genes encoding regulatory proteins of the alternative complement pathway and of complement C3 (C3) have been identiﬁed as concausative factors. Methods: Three children with MPGN type I, four with C3G, i.e. three with C3 glomerulonephritis (C3GN) and one with dense deposit disease (DDD), were followed. Clinical, autoimmune data, histological characteristics, estimated glomerular ﬁltration rate (eGFR), proteinuria, serum C3, genetic and biochemical analysis were assessed. Results: The median age at onset was 7.3 years and the median eGFR was 72 mL/min/1.73 m . Six children had marked proteinuria. All were treated with renin–angiotensin–aldosterone system (RAAS) blockers. Three were given one or more immunosuppressive drugs and two eculizumab. At the last median follow-up of 9 years after diagnosis, three children had normal eGFR and no or mild proteinuria on RAAS blockers only. Among four patients without remission of proteinuria, genetic analysis revealed mutations in complement regulator proteins of the alternative pathway. None of the three patients with immunosuppressive treatment achieved partial or complete remission of proteinuria and two progressed to ESRD and renal transplantation. Two patients treated with eculizumab revealed relevant decreases in proteinuria. Received: 31.8.2017. Editorial decision: 27.12.2017 V C The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 2| G. Sparta ` et al. Conclusions: In children with MPGN type I and C3G, the outcomes of renal function and response to treatment modality show great variability independent from histological diagnosis at disease onset. In case of severe clinical presentation at disease onset, early genetic and biochemical analysis of the alternative pathway dysregulation is recommended. Treatment with eculizumab appears to be an option to slow disease progression in single cases. Key words: C3 glomerulopathy, complement dysregulation, eculizumab, MPGN, paediatrics and C3G is based on single-centre studies and expert opinions. Introduction Clinical trials in adults and children with different treatment Membranoproliferative glomerulonephritis (MPGN) with modality propositions [e.g. immunosuppressant agents, anti- immune complexes is a rare chronic glomerulonephritis in platelet drugs and plasmapheresis (PEX)] are described [23–29]. childhood characterized by proteinuria (up to the nephrotic Treatment with renin–angiotensin–aldosterone system (RAAS) range), haematuria, hypertension and often impaired renal blockers are described to induce a decrease in proteinuria and function at disease onset . In up to 50% of affected children, delays progression to ESRD in many glomerular diseases in MPGN leads to renal failure within 10 years . Impaired renal adults [30, 31] and in some glomerulopathies in children [32, 33]; function after 1 year of onset is considered a risk factor for poor however, there is no evidence for beneficial effect in children renal outcome and end-stage renal disease (ESRD) . The with MPGN I  and spontaneous recovery in C3GN can also recurrence rate after renal transplantation (RTPL) is high (up to not be excluded . 45%) [1–4]. MPGN may occur as a primary genetic disorder or Eculizumab is a monoclonal antibody binding to C5 and secondary to chronic diseases, including infections (e.g. hepati- thereby inhibiting the complement system and preventing acti- tis B or C), systemic lupus erythematosus, liver disease and vation of the alternative pathway. Recently, promising results malignancies. were shown in selected patients with MPGN and C3GN charac- In the past, MPGN was diagnosed and classified by renal his- terized by the presence of alterations in regulatory proteins of tological features and grouped into three pathological subtypes the alternative complement pathway [22, 35–37]. with different aetiologies and pathogenesis, types I, II [(dense We evaluated the outcome of seven children with the diag- deposit disease (DDD)] and III . Activation of the alternative nosis of MPGN I, C3GN and DDD. All were screened for the pres- complement pathway has repeatedly been observed in conjunc- ence of genetic mutations of the alternative complement tion with low serum levels of complement C3 (C3) [6–8]. A link pathway. We analysed the benefit of different administered between dysregulation of the alternative complement pathway treatments. and the pathogenesis of MPGN was assumed  and has recently been confirmed by findings of mutations in the genes Materials and methods of complement factor H (CFH) and CF-related proteins (CFHR) in DDD [9–13]. All patients with histologically diagnosed MPGN I, DDD and Therefore the histological classification has been reconsid- C3GN who were seen at the University Children’s Hospital ered recently on the basis of pathogenesis and with division Zurich between 2003 and 2015 were included in a retrospective into those cases in which the glomerular immune deposits stain data analysis. Clinical assessment included diagnosis, sex, med- for immunoglobulins and complement and those cases that are ical history, clinical examination, blood pressure (BP) and analy- characterized by C3 deposition alone [5, 14–16]. The term ‘C3 sis to exclude potentially secondary forms of MPGN. glomerulopathy’ (C3G) encompassed complement-mediated renal disease, and therefore incorporates disease entities where Biochemical and genetic analysis the presence of a disease-associated complement mutation is causally associated with the underlying renal pathology. Plasma creatinine, total serum protein and albumin and urinary Examples include DDD and C3 glomerulonephritis (C3GN) . protein:creatinine ratio (UPCR) were analysed at disease onset The term C3GN was coined to describe glomerular lesions in and at the last follow-up. Blood samples for complement regu- which there is glomerular accumulation of C3 with little or no lator protein analysis were centrifuged immediately after col- immunoglobulin in the absence of the characteristic highly lection and frozen at80 C prior to analysis. C3 serum levels electron-dense transformation seen in DDD . The incidence were evaluated by kinetic nephelometry (Immage 800, Beckman of C3G is estimated to be 1–2 per 10 children [17, 18], with dis- Coulter, Brea, CA, USA) with a normal value>0.7 g/L (reference ease recurrence after RTPL reported at between 30 and 77% and value of the immunology laboratory of Zurich University a graft failure due to recurrence in 17–50% of the recipients Children’s Hospital). The soluble complement components C5b- [19, 20]. MPGN associated with the presence of immunoglobu- 9 (membrane attack complex), C3d and C3 nephritic factor lins and complement has been termed immune complex–medi- (C3NeF) were measured from plasma samples at the Institute ated MPGN by Sethi and Fervenza . Immune complex- for Immunology of Heidelberg University (Heidelberg, mediated MPGN is commonly associated with autoimmune dis- Germany). The following biochemical analyses were performed ease and chronic infection and can be associated with mixed at follow-up from plasma and serum, as well as ethylenediami- cryoglobulinaemia or monoclonal gammopathy [5, 21]. These netetraacetic acid blood samples for genetic analysis (samples associations were excluded in our study. In our series, histologi- at the Leibniz Institute for Natural Product Research and cal MPGN I is a case of so-called idiopathic MPGN. MPGN I and Infection Biology, Jena, Germany): CFH antibody, C3-convertase C3G are regarded as heterogeneous diseases, with several stud- antibody, CFI, CFB, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, MCP ies reporting complement mutations in complement genes [15, (CD46) and by next-generation sequencing (NGS) at the Center 22]. So far there are no evidence-based guidelines for treatment for Human Genetics at Bioscientia in Ingelheim (Germany), as of MPGN I [1, 2] and C3G. The mainstay of treatment in MPGN I described in detail below. Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 MPGN and C3 glomerulopathy in children | 3 Targeted NGS using a customized multigene panel for atypi- the native kidneys and one in the transplanted kidney. The cal hemolytic uremic syndrome (aHUS) and related disorders biopsy diagnosis was based on the current classification and was performed in all patients . In brief, we utilized a custom- nomenclature for C3G and MPGN [5, 16] on findings by light ized sequence capture library that targets exons and addition- microscopy (LM), immunofluorescence (IF) and electron micro- ally 35 bp of flanking intronic sequence (20–23). Genomic DNA scopy (EM). was fragmented and the coding exons of the analysed genes, as well as the corresponding exon–intron boundaries, were enriched using the Roche/NimbleGen sequence capture Definitions approach (NimbleGen, Madison, WI, USA), amplified and sequenced simultaneously by Illumina NGS sequencing-by- The following definitions were used: nephrotic-range proteinuria: synthesis technology using a HiSeq 1500 system (Illumina, San UPCR >250 g/mol; nephrotic syndrome: serum albumin<25 g/L, Diego, CA, USA). Target regions were usually sequenced with an nephrotic-range proteinuria and generalized oedema; remission: average coverage of 400–500-fold. With this method, 20-fold UPCR <20 g/mol; partial remission: UPCR >20–<80 g/mol; normal coverage is obtained for >99.5% of the regions of interest. NGS renal function: estimated glomerular filtration rate data analysis was performed using bioinformatic analysis tools (eGFR)>90 mL/min/1.73 m , as calculated by the Schwartz and JSI Medical Systems software (version 4.1.2; JSI Medical formula using a local k-factor of 40 ; hypertension: casual sys- Systems, Kippenheim, Germany). Identified variants and indels tolic BP >95th percentile for sex and height . were filtered against external and internal databases and depending on allelic frequency. The focus was on rare variants with a minor allele frequency of 1%. Nonsense, frameshift and canonical splice site variants were considered a priori as Treatment likely to be pathogenic. Pathogenicity of identified non- All patients were given RAAS blockers as first-line antiproteinu- synonymous variants was assessed using bioinformatic predic- ric therapy. Additional treatment consisted of prednisolone tion programs, such as Mutation Taster, Polyphen-2, (PDN), in accordance with the dosage regimen for treatment of MutationAssessor and FATHMM. Only those variants predicted children with steroid-resistant nephrotic syndrome [41, 42]. by the majority of algorithms used to be probably damaging Further immunosuppressive treatment [cyclosporine A (CSA) were considered likely to be pathogenic. In silico analysis of and mycofenolate mofetile (MMF)] [25, 42] was administered to splice site effects was performed using bioinformatic programs patients who failed to attain partial or full remission of protei- such as Fruitfly, NetGene2, Human Splicing Finder, Mutation nuria. Eculizumab has been available in our hospital since 2013, Taster and ESEFinder. Mapping and coverage statistics were but costs were not covered by all insurances, as this drug is still generated from the mapping output files using GATK. The off-label for MPGN/C3G treatment. Eculizumab was given to two resulting sequence data were compared with the reference patients with nephrotic-range proteinuria despite immunosup- sequence of the RefSeq database. High coverage enabled copy pressive and RAAS blocker treatment. The dosage followed cur- number variation analysis. Potential copy number alterations rent recommendations for aHUS . Patients treated with were initially identified by VarScan on mapped reads. In this eculizumab were immunized with meningococcal vaccine way, coverage of every target region of the sample was inter- nally normalized and compared with normalized control data (Menveo, GlaxoSmithKline Biologicals, Rixensart, Belgium) of other samples of the same run by VarScan copy number 4 weeks prior to administration. Informed consent was obtained mode and standard settings. Putative pathogenic differences from all parents of included patients for medical data collection, between the wild-type sequence (human reference genome genetic analysis and treatment with eculizumab. according to the University of California, Santa Cruz Genome Browser: hg19, GRCh37) and the patient’s sequence were vali- dated by conventional Sanger sequencing and, in the case of copy number variation, by multiplex ligation-dependent probe Results amplification. Seven children (four boys and three girls) fulfilled the inclusion criteria (Table 1): three patients suffered from MPGN I, three from Renal biopsy C3GN and one from DDD. The median age at onset was 7.3 years (range 2.5–12.5) with a median eGFR of 72 mL/min/1.73 m (range All patients underwent native kidney biopsy at disease onset. Repeated renal biopsy was performed in two children, one of 41–140) at diagnosis. Table 1. Patient characteristics at disease onset Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Age (years) 7.3 2.5 6 12.5 5.9 10.5 8.8 Sex Male Male Female Female Male Female Male Hypertension No No No No No Yes Yes Haematuria Micro Micro Macro Micro Macro Micro Micro UPCR (g/mol) (reference <20) 634 126 530 855 580 1500 1000 Nephrotic syndrome (UPCR >250 g/mol) No No No Yes No Yes Yes Serum albumin (g/L) (reference 35–51) 27 32 26 11 30 12 12 eGFR (mL/min/1.73 m ) (reference >90) 72 140 41 67 88 65 95 Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 4| G. Sparta ` et al. Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 Table 2. Genetic, complement and histology testing Test Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Genetic testing CFHR1 CFHR1 c.880G>A (p.Glu294Lys) het; CFHR1/3 del het CFHR1/3 del het CFHR1/3 del het CFHR2 CFHR2 c.109G>A (p.E37K) het; c.584G>C(pG195A) CFHR5 CFHR5: c970þ 2T>G het het CFH-het (¼two functional Factor H polymorphisms) MCP: c.989– MCP: het risk haplo- MCP: het risk MCP: het risk MCP: het risk MCP: hom risk MCP: het risk MCP 78G>A hom, type MCP-H1 haplotype haplotype haplotype haplotype MCP-H1 haplotype MCP- c.*897T>C hom MCPggaac MCPggaac MCP-H1 H1 (hom risk haplotype MCPggaac) C3 antibody positivity No No No No No Yes Yes a a C3NeF Negative Negative Negative Negative Positive Negative Negative C3 g/L (reference 0.4 <0.06 0.23 0.26 0.3 0.24 0.6 0.7–1.76) Renal histology MPGN I MPGN I C3 GN MPGN I C3-GN C3-GN DDD biopsy LM Mesangial and Mesangial and endo- Mesangial and mild Mesangial and mild Mesangial and mild Mesangial and endo- Mesangial and endocapillary capillary prolifera- endocapillary pro- endocapillary pro- endocapillary capillary prolifera- mild endoca- proliferation. tion. Double liferation, double liferation, double proliferation tion, double pillary prolifer- Double con- contours of GBM contours of GBM, contours of GBM, contours of GBM, ation, 25% tours of GBM humps crescents, 36% glo- 40% hyalinized hyalinized glo- meruli hyalinized, glomeruli, seg- meruli, cres- 50% interstitial ﬁb- mental sclerosis, cents in 50% of rosis and tubular crescents, >95% glomeruli, atrophy. interstitial ﬁbrosis thickended, and tubular glassy GBM atrophy IF Dominant GBM Dominant GBM and GBM and mesangial Dominant GBM and Dominant mesangial Mesangial and GBM GBM positivity and mesangial mesangial positiv- positivity for C3 mesangial positiv- and GBM positiv- positivity for C3 for C3 (3þ), IgM positivity for ity for C3 (3þ), IgG (3þ), IgM (1þ). IgG ity for IgG (3þ) and ity for C3 (3þ). IgG (3þ), less IgG (1þ) (3þ), IgG (1þ) IgG (2þ) and C3 (2þ), IgM (3þ) is negative C3 (3þ) is negative (2þ) Electron Subendothelial Subendothelial, Mesangial, subendo- Mesangial, subendo- Granular, not very Mesangial and intra- Highly osmio- microscopy and mesangial mesangial and thelial, intramem- thelial, intramem- dense intramem- membranous, not philic segmen- electron-dense rare small subepi- branous and branous and rare branous and very dense granu- tal electron- deposits thelial and intra- subepithelial elec- subepithelial elec- mesangial elec- lar electron-dense dense deposits membranous elec- tron-dense depos- tron-dense tron-dense deposits. Rare in lamina tron-dense its with humps deposits deposits small subepithe- densa of GBM deposits lial deposits. a b Hom, homozygous; het, heterozygous; del, deletion; GBM, glomerular basement membrane. Result performed only under treatment with eculizumab in Patient 5 and with PEX in Patient 7. Values of serum complement C3 at dis- ease onset. Second biopsy 7 years after onset biopsy. MPGN and C3 glomerulopathy in children | 5 Analysis of the alternative complement pathway: CFs proteinuria on PDN and were given additional immunosuppres- sive treatment. and genetic testing All children showed persistently low serum C3 (Table 2). In Patients with RAAS blockers only addition, C3NeF was negative in all but one patient and C3b antibodies were positive in two children with C3GN. Patients 1–3 (MPGN I, n5 2; C3GN, n5 1) (Table 3). All had nor- At the last follow-up (Tables 3–5), C3d was elevated in five mal renal function at onset without signs of a nephrotic syn- patients and sC5b-9 levels were increased in four children, both drome. Follow-up was uneventful, with partial or complete in MPGN I and C3GN. remission of proteinuria and maintained normal renal function. All had persistently low C3. Genetic testing revealed a risk hap- Renal biopsy lotype for the membrane cofactor protein (MCP/CD46) gene in all three children. In addition, Patient 2 (MPGN I) had elevated Renal biopsy findings (MPGN I, C3GN and DDD) (Figures 1–4) are sC5b-9 and a heterozygous mutation in the CFHR5 gene. presented in Table 2. Repeated renal biopsy was performed in Patient 4 (native kidney, before embarking on MMF) and Patient 7 (renal graft for suspected acute graft rejection). The diagnoses Patients with additional immunosuppressive were made based on IF findings. As the IF slides were no longer treatments, including eculizumab available, the figures contain pictures of immunohistochemis- Patient 4 (MPGN I) (Tables 4). Because of progressive proteinuria try performed for the purpose of the publication. on PDN, MMF was started . Daily proteinuria initially decreased from 4 to 2 g. However, impaired renal function and Treatment regimens and follow-up nephrotic-range proteinuria persisted with elevated alternative The median follow-up was 9 years (range 2.2–11.5). Five children pathway activity. A second renal biopsy confirmed the diagno- maintained renal function of their native kidneys, with a sis of MPGN I. Therefore MMF was discontinued and eculizumab median eGFR of 107 mL/min/1.73 m (range 40–180), including was started. Six months after starting eculizumab, a significant four patients with normal eGFR. Two children reached ESRD decrease in proteinuria and sC5b-9 was observed with stabiliza- and underwent RTPL with initially good renal graft function. All tion of eGRF. children had BP < 95th percentile, with six on RAAS blockers: in one child, RAAS blockers were stopped after RTPL. All children Patient 5 (C3GN) (Tables 4). Because of increasing proteinuria exhibited persistent haematuria. Three patients with nephrotic with normal renal function and persistently elevated alterna- syndrome (Patients 4, 6 and 7) exhibited progression of tive pathway activity despite RAAS blockade, the treatment Fig. 1. DDD native kidney (Patient 7). (A) Glomeruli with mesangial and endocapillary proliferation and a ﬁbrocellular crescent [hematoxylin and eosin (H&E) stain, orig- inal magniﬁcation 200]. (B) Intramembranous and mesangial deposits (acid fuchsin orange G stain, original magniﬁcation 200). (C and D) EM with highly osmiophilic electron-dense deposits in the lamina densa of the glomerular basement membrane and mesangium (original magniﬁcation 1100 and 1950). Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 6| G. Sparta ` et al. Fig. 2. DDD recurrence in transplant biopsy (Patient 7). (A) Thickened, glassy GBM and endocapillary hypercellularity with mononuclear cells (H&E stain, original mag- niﬁcation 200). (B) Intramembranous deposits and endocapillary proliferation (AFOG stain, original magniﬁcation 324). (C) Immunohistochemistry positive for C3 (original magniﬁcation 200). (D) Immunohistochemistry negative for immunoglobulin G (original magniﬁcation 200). (E and F) EM with highly osmiophilic electron- dense deposits in the lamina densa and subepithelial humps (original magniﬁcation 1000 and 2500). with eculizumab was initiated . After 11 months, proteinuria Seven months after transplantation, sC5b-9 was still slightly and sC5b-9 level decreased, whereas C3NeF remained positive. elevated. Renal graft function remained stable without Renal function remained stable at a normal level. proteinuria. Patient 6 (C3GN) (Table 5). Nephrotic-range proteinuria per- Patient 7 (DDD) (Table 5). Additional treatment with CSA was sisted despite additional treatment with CSA for 6 years. The started due to persistent nephrotic-range proteinuria. The child child reached ESRD and underwent peritoneal dialysis 6.5 years reached ESRD 4.2 years after disease onset. Living paternal-donor after disease onset. Deceased-donor RTPL was performed; RTPL was performed; immunosuppression included CSA, MMF immunosuppression included tacrolimus, MMF, PDN and induc- and PDN. The child experienced acute/active antibody-mediated tion therapy with basiliximab. RAAS blocker was stopped. rejection (aABMR) and recurrence of DDD in the graft 3.3 years Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 MPGN and C3 glomerulopathy in children | 7 Fig. 3. MPGN I (Patient 1). (A) Mesangial and endocapillary proliferation (periodic acid–Schiff stain, original magniﬁcation 110). (B) Splitting of the GBM (silver methen- amine stain, original magniﬁcation 220). (C) Subendothelial deposits (AFOG stain, original magniﬁcation 200). (D) Immunhistochemistry positive for IgG (original magniﬁcation 280), inset: positivity for C3 (original magniﬁcation 220). (E) EM with mostly subendothelial and some subepithelial and mesangial electron-dense deposits (original magniﬁcation 1950). (F) Subendothelial and small subepithelial electron-dense deposits (original magniﬁcation 10 500). after transplantation Therefore treatment with CSA was switched showed normal values. Renal graft function was impaired (eGFR to tacrolimus and PEX was started [1.5-fold plasma volume 42 mL/min/1.73 m ) at the last follow-up. exchange with fresh frozen plasma (FFP)], but ESRD occurred 3.9 years after RTPL. A deceased-donor RTPL was then performed. Discussion Immunosuppression included tacrolimus, MMF, PDN and induc- tion with thymeoglobulin. Again, ABMR and recurrence of the dis- MPGN and C3G are rare diseases with chronic progressive glo- ease with nephrotic proteinuria occurred 15 months after merulonephritis and children may have an unfavourable course retransplantation. Weekly treatment with PEX reduced proteinu- leading to ESRD [17, 18, 44, 45]. Studies in adults and children ria, but proteinuria increased when the PEX interval was extended have shown that proteinuria is a major risk factor for develop- to every second week. Measurement of sC5b-9 (after PEX session) ing ESRD [1, 46, 47]. In the last decade, new insights are Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 8| G. Sparta ` et al. Fig. 4. C3GN (Patient 6). (A) Mesangial and endocapillary proliferation (H&E stain, original magniﬁcation 100). (B) Mesangial and GBM deposits (AFOG stain, original magniﬁcation 200). (C) Immunohistochemistry positive for C3 (original magniﬁcation 200). (D) Immunohistochemistry negative for IgG (original magniﬁcation 200). (E) EM with mostly intramembranous and mesangial not very dense electron-dense deposits (original magniﬁcation 300). (F) EM with subepithelial, intramem- branous and subendothelial deposits (original magniﬁcation 2000). emerging to improve genetic and biochemical investigations of alternative pathway, has shown promising results in the treatment these diseases. of some cases of MPGN and C3G [22, 35–37, 50]. Currently there is no established treatment for MPGN and C3G. Our analysis revealed a dysregulation of the complement Patients appear to respond differently to various therapy modal- alternative pathway and mutation/variation in genes of CF pro- ities [27, 29, 48, 49]. The majority of treatment regimens and case teins in children with MPGN, C3GN and DDD. Three patients of series have been reported in adults, not in children [23, 26], and are our series showed a favourable outcome. These were two often associated with significant side effects. Recently, treatment patients with MPGN I and one with C3GN, the latter without any with eculizumab, a monoclonal antibody binding to C5 of the genetic variation in CFHR proteins. Five of seven children had a Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 MPGN and C3 glomerulopathy in children | 9 Table 3. Patients with RAAS blockers only: values at last follow-up Characteristic Patient 1 Patient 2 Patient 3 Maintained renal function Native kidney Native kidney Native kidney Treatment Enalapril Enalapril Enalapril Losartan Losartan Duration of observation (years) 9.7 2.2 4.8 eGFR 143 97 180 C3 (g/L) (reference 0.7–1.76) 0.6 <0.06 0.13 UPCR (g/mol) (reference < 20) 41 41 <20 sC3d (mU/L) (reference < 40) 50 103 35 sC5b-9 (ng/mL) (reference < 320) 239 2538 164 eGFR rate according to Schwartz formula in mL/min/1.73 m . Table 4. Patients with eculizumab: values before eculizumab and at spontaneous remission may not be excluded . Consistent last follow-up with other authors , those children of our series with neph- rotic syndrome at onset showed a more severe course, with two Patient 4 Patient 5 progressing to ESRD followed by RTPL. Characteristic (original disease) (MPGN I) (C3GN) Eculizumab has been described as a successful treatment of patients with MPGN [35, 50, 53–55] and C3G [19, 22, 56] in several Maintained renal function Native kidney Native kidney Additional treatments to PDN reports. Elevated sCb5-9 levels may be a predictor of response to RAAS blockers MMF treatment with eculizumab, but other factors affecting response Before eculizumab to therapy are poorly understood [53, 55]. Two patients of our Duration of observation (years) 6.9 9.5 series, one with C3GN and one with MPGN I, exhibited a signifi- eGFR (mL/min/1.73 m ) 40 100 cant decrease in proteinuria on eculizumab in their native kid- C3 (g/L) (reference 0.7–1.76) 0.09 0.06 neys, but elevated activity of the alternative pathway persisted. UPCR (g/mol) (reference <20) 510 750 This is consistent with observations of other paediatric patients sC3d (mU/L) (reference <40) 63 95 with MPGN  suggesting that eculizumab is not completely sC5b-9 (ng/mL) 4100 6500 effective in suppressing sC5b-9 activity in C3G. They hypothe- (reference >320) sized that sC5b-9 alone may not reflect disease activity. The Observation on eculizumab presence of mutations alone does not significantly increase the Duration of therapy (months) 6 11 risk of developing idiopathic MPGN or C3G, but they do so when eGFR 45 90 combined with common susceptibility variants [i.e. in CD46, UPCR (g/mol) 270 227 CFH or Thrombomodulin (THBD)] [17, 52]. sC5b-9 (ng/mL) 282 639 C3NeF, an auto-antibody directed against the alternative C3- C3 (g/L) 0.12 0.14 convertase, was positive at follow-up in only one child of our series. Published reports on the impact of C3NeF on outcome in C3G patients are inconsistent, with reported patients ranging from complete remission to ESRD [57, 58]. Apparently C3NeF can fluctuate during the clinical course independent of the dis- heterozygous mutation/deletion or variation in CFHR proteins 1, ease treatment . Others observed a higher risk of progressing 2, 3 or 5. Recent findings show that dysregulation of the comple- to ESRD in patients without complement gene mutation or ment pathway by CFHR2–CFHR5 hybrid protein leads to C3NeF, stabilizing the alternative pathway C3-convertase . enhanced C3-convertase activation of the alternative comple- In our patient, C3NeF and a heterozygous variation of CFHR1 ment pathway and other genetic complement abnormalities and CFHR1/3 was found. In addition, sC5b-9 was significantly associated with MPGN [12, 17, 51]. In our patients, genetic alter- elevated. Other treatments, for example, rituximab and PEX, are ations, including variation or polymorphisms, may at least in controversially discussed in the literature with different results part explain the different outcomes and responses to the vari- [52, 58, 59]. Therefore, after a risk–benefit evaluation of these ous treatment modalities. Similar findings are described by treatment options, we decided to apply eculizumab in our other authors, where the clinical presentation and the measure- young child. The outcome was favourable, with stable, normal ment of plasma C3, C3d and sC5b-9 do not allow differentiation renal function and a significant decrease in proteinuria between C3G and MPGN I . Recent knowledge suggests that 11 months after eculizumab treatment was started. in idiopathic MPGN—an immune complex Rixensart, Belgium In a patient undergoing RTPL twice, C3NeF and sC5b-9 were mediated disease—involvement of the alternative pathway analysed after PEX treatment with FFP had been started, so plays an important role [17, 52]. detection of both C3NeF and sC5b-9 might have been missed Three of seven of our patients (two with MPGN and one with . This patient experienced aABMR with recurrence of DDD 3 C3GN) without nephrotic syndrome showed remission of protei- years after the first RTPL and was therefore treated with PEX. nuria on RAAS blocker therapy only. No significant reduction in However, he reached ESRD after the first RTPL, with recurrence proteinuria is described in children with MPGN treated with of the disease in the second renal graft. Treatment with PEX RAAS blocker only. Other data revealed that patients with less was started at weekly intervals but led to only a moderate parenchymal damage in their initial renal biopsy benefit the decrease in proteinuria with persistently impaired graft func- most from sole treatment with RAAS blocker ; however, tion. The insurance refused to pay for treatment with Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 10 | G. Sparta ` et al. Table 5. Patients undergoing RTPL: values before RTPL and at last follow-up after RTPL Characteristic (original disease) Patient 6 (C3GN) Patient 7 (DDD) Before RTPL Additional treatments to RAAS blockers CSA CSA Duration of observation (years) 9 11.5 C3 (g/L) (reference 0.7–1.76) 0.2 0.55 UPCR (g/mol) (reference <20) 1350 400 sC3d (mU/L) (reference <40) 27 61 sC5b-9 (ng/mL) (reference <320) 1359 Not done Observation after RTPL (m) Duration (months) 7 48 (2nd RTPL) Recurrence of original disease (m) No Yes (15) Treatment for original disease recurrence No PEX/FFP Graft rejection No Yes (ABMR) eGFR 51 42 UPCR (g/mol) <20 400 sC5b-9 (ng/mL) 770 121 C3 (g/L) 0.25 0.9 Renal graft biopsy after RTPL No Yes Treatment after RTPL Induction with basiliximab for RTPL Induction with thymeoglobulin for RTPL Prednisolone Prednisolone Tacrolimus Tacrolimus MMF MMF 3. Ponticelli C, Glassock RJ. Posttransplant recurrence of primary eculizumab. As complement-mediated dysregulation is involved in the pathophysiology of aABMR , it is conceivable glomerulonephritis. Clin J Am Soc Nephrol 2010; 5: 2363–2372 that DDD recurrence may have triggered aABMR by inducing 4. Alasfar S, Carter-Monroe N, Rosenberg AZ et al. complement activation. In a recent study, MPGN recurrence Membranoproliferative glomerulonephritis recurrence after kidney transplantation: using the new classiﬁcation. BMC after RTPL was detected in 18 of 40 transplants and in 3 cases Nephrol 2016; 17: 7 disease recurrence preceded aABMR and led to graft loss . 5. Cook HT, Pickering MC. Histopathology of MPGN and C3 glo- Registries have also reported recurrence rates on renal graft for merulopathies. Nat Rev Nephrol 2015; 11: 14–22 DDD and C3G of 50% and 43–67%, respectively . 6. West CD. Idiopathic membranoproliferative glomerulo- There are some limitations to our study: (i) its retrospective nephritis in childhood. Pediatr Nephrol 1992; 6: 96–103 character; (ii) the individual treatment approach based on the 7. Daha MR, Fearon DT, Austen KF. Formation in the presence of clinical course; (iii) due to the different time period at disease C3 nephritic factor (C3NeF) of an alternative pathway C3 con- manifestation of each patient, no uniformity of treatment was vertase containing uncleaved B. Immunology 1976; 31: 789–796 possible and (iv) measurement of activity of the alternative 8. Licht C, Schlotzer-Schrehardt U, Kirschﬁnk M et al. MPGN II – pathway (i.e. C3d, sC5b-9) and C3NeF was not available at dis- genetically determined by defective complement regula- ease onset. tion? Pediatr Nephrol 2007; 22: 2–9 Our results reveal that early examination of the alternative 9. Licht C, Heinen S, Jozsi M et al. Deletion of Lys224 in regula- complement pathway may aid to define a more individually tory domain 4 of factor H reveals a novel pathomechanism tailored treatment. However, we observed heterogeneity of clin- for dense deposit disease (MPGN II). Kidney Int 2006; 70: 42–50 ical and biological features in MPGN and C3G and therefore the 10. Skerka C, Lauer N, Weinberger AA et al. Defective comple- difficulty of interpretation of both genetic abnormalities and ment control of factor H (Y402H) and FHL-1 in age-related biochemical analysis. Long-term observation is necessary in macular degeneration. Mol Immunol 2007; 44: 3398–3406 order to draw conclusions about the results of treatments and 11. Dragon-Durey MA, Fremeaux-Bacchi V, Loirat C et al. renal function at follow-up. Heterozygous and homozygous factor h deﬁciencies associ- ated with hemolytic uremic syndrome or membranoproli- Conflict of interest statement ferative glomerulonephritis: report and genetic analysis of 16 cases. J Am Soc Nephrol 2004; 15: 787–795 None declared. 12. Chen Q, Wiesener M, Eberhardt HU et al. Complement factor H-related hybrid protein deregulates complement in dense References deposit disease. J Clin Invest 2014; 124: 145–155 1. Cansick JC, Lennon R, Cummins CL et al. Prognosis, treat- 13. Chen Q, Manzke M, Hartmann A et al. Complement factor H- ment and outcome of childhood mesangiocapillary (mem- related 5-hybrid proteins anchor properdin and activate branoproliferative) glomerulonephritis. Nephrol Dial complement at self-surfaces. J Am Soc Nephrol 2016; 27: Transplant 2004; 19: 2769–2777 1413–1425 2. Smith RJ, Alexander J, Barlow PN et al. New approaches to 14. Walker PD, Ferrario F, Joh K et al. Dense deposit disease is the treatment of dense deposit disease. J Am Soc Nephrol not a membranoproliferative glomerulonephritis. Mod Pathol 2007; 18: 2447–2456 2007; 20: 605–616 Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 MPGN and C3 glomerulopathy in children | 11 36. Daina E, Noris M, Remuzzi G. Eculizumab in a patient with 15. Sethi S, Nester CM, Smith RJ. Membranoproliferative glomer- ulonephritis and C3 glomerulopathy: resolving the confu- dense-deposit disease. N Engl J Med 2012; 366: 1161–1163 sion. Kidney Int 2012; 81: 434–441 37. McCaughan JA, O’Rourke DM, Courtney AE. Recurrent dense 16. Pickering MC, D’Agati VD, Nester CM et al. C3 glomerulop- deposit disease after renal transplantation: an emerging role athy: consensus report. Kidney Int 2013; 84: 1079–1089 for complementary therapies. Am J Transplant 2012; 12: 17. Servais A, Noe ¨ l L-H, Roumenina LT et al. Acquired and 1046–1051 genetic complement abnormalities play a critical role in 38. Bachmann NGM, Hiersche M, Ha ¨ ffner K et al. Comprehensive dense deposit disease and other C3 glomerulopathies. genetic analysis by next-generation sequencing (NGS) in a Kidney Int 2012; 82: 454–464 cohort of 264 patients with aHUS. Oral presentation at the 18. Medjeral-Thomas N, Malik TH, Patel MP et al. A novel CFHR5 5th International Conference on HUS & Related Disorders, fusion protein causes C3 glomerulopathy in a family without Innsbruck, Austria, 2015. http://www.hus-online.at/de/ Cypriot ancestry. Kidney Int 2014; 85: 933–937 Conference2_en.html 19. Lebreton C, Bacchetta J, Dijoud F et al. C3 glomerulopathy 39. Schwartz GJ, Munoz A, Schneider MF et al. New equations to and eculizumab: a report on four paediatric cases. Pediatr estimate GFR in children with CKD. J Am Soc Nephrol 2009; 20: Nephrol 2017; 32: 1023–1028 629–637 20. Bacchetta J, Cochat P. Primary disease recurrence-effects on 40. Update on the 1987 Task Force Report on High Blood paediatric renal transplantation outcomes. Nat Rev Nephrol Pressure in Children and Adolescents: a working group 2015; 11: 371–384 report from the National High Blood Pressure Education 21. Sethi S, Fervenza FC. Membranoproliferative Program. National High Blood Pressure Education Program glomerulonephritis–a new look at an old entity. N Engl J Med Working Group on Hypertension Control in Children and 2012; 366: 1119–1131 Adolescents. Pediatrics 1996; 98: 649–658 22. Riedl M, Thorner P, Licht C. C3 Glomerulopathy. Pediatr 41. Ehrich JH, Brodehl J, Arbeitsgemeinschaft fur Padiatrische Nephrol 2017; 32: 43–57 Nephrologie. Long versus standard prednisone therapy for 23. Levin A. Management of membranoproliferative glomerulo- initial treatment of idiopathic nephrotic syndrome in chil- nephritis: evidence-based recommendations. Kidney Int dren. Eur J Pediatr 1993; 152: 357–361 1999; 55 (Suppl 70) S41–S46 42. Lombel RM, Gipson DS, Hodson EM et al. Treatment of 24. Somers M, Kertesz S, Rosen S et al. Non-nephrotic children steroid-sensitive nephrotic syndrome: new guidelines from with membranoproliferative glomerulonephritis: are ste- KDIGO. Pediatr Nephrol 2013; 28: 415–426 roids indicated? Pediatr Nephrol 1995; 9: 140–144 43. Schmidtko J, Peine S, El-Housseini Y et al. Treatment of atyp- 25. De S, Al-Nabhani D, Thorner P et al. Remission of resistant ical hemolytic uremic syndrome and thrombotic microan- MPGN type I with mycophenolate mofetil and steroids. giopathies: a focus on eculizumab. Am J Kidney Dis 2013; 61: Pediatr Nephrol 2009; 24: 597–600 289–299 26. Jones G, Juszczak M, Kingdon E et al. Treatment of idiopathic 44. Ault BH. Factor H and the pathogenesis of renal diseases. membranoproliferative glomerulonephritis with mycophe- Pediatr Nephrol 2000; 14: 1045–1053 nolate mofetil and steroids. Nephrol Dial Transplant 2004; 19: 45. Schwertz R, de Jong R, Gretz N et al. Outcome of idiopathic 3160–3164 membranoproliferative glomerulonephritis in children. Acta 27. Bagheri N, Nemati E, Rahbar K et al. Cyclosporine in the Paediatr 1996; 85: 308–312 treatment of membranoproliferative glomerulonephritis. 46. Warady BA, Abraham AG, Schwartz GJ et al. Predictors of Arch Iran Med 2008; 11: 26–29 rapid progression of glomerular and nonglomerular kidney 28. Appel GB, Cook HT, Hageman G et al. Membranoproliferative disease in children and adolescents: the chronic kidney dis- glomerulonephritis type II (dense deposit disease): an ease in children (CKiD) cohort. Am J Kidney Dis 2015; 65: update. J Am Soc Nephrol 2005; 16: 1392–1403 878–888 29. Rabasco C, Cavero T, Roman E et al. Effectiveness of myco- 47. Sandsmark DK, Messe SR, Zhang X et al. Proteinuria, but not phenolate mofetil in C3 glomerulonephritis. Kidney Int 2015; eGFR, predicts stroke risk in chronic kidney disease: chronic 88: 1153–1160 renal insufﬁciency cohort study. Stroke 2015; 46: 2075–2080 30. Ruggenenti P, Perna A, Gherardi G et al. Renoprotective prop- 48. Nester CM, Smith RJ. Treatment options for C3 glomerulop- erties of ACE-inhibition in non-diabetic nephropathies with athy. Curr Opin Nephrol Hypertens 2013; 22: 231–237 non-nephrotic proteinuria. Lancet 1999; 354: 359–364 49. Nasr SH, Valeri AM, Appel GB et al. Dense deposit disease: 31. Brenner BM, Cooper ME, de Zeeuw D et al. Effects of losartan clinicopathologic study of 32 pediatric and adult patients. on renal and cardiovascular outcomes in patients with type Clin J Am Soc Nephrol 2009; 4: 22–32 2 diabetes and nephropathy. N Engl J Med 2001; 345: 861–869 50. Oosterveld MJ, Garrelfs MR, Hoppe B et al. Eculizumab in 32. Yang Y, Ohta K, Shimizu M et al. Treatment with low-dose pediatric dense deposit disease. Clin J Am Soc Nephrol 2015; angiotensin-converting enzyme inhibitor (ACEI) plus angio- 10: 1773–1782 tensin II receptor blocker (ARB) in pediatric patients with IgA 51. Abrera-Abeleda MA, Nishimura C, Smith JL et al. Variations nephropathy. Clin Nephrol 2005; 64: 35–40 in the complement regulatory genes factor H (CFH) and fac- 33. Camacho Diaz JA, Gimenez Llort A, Garcia Garcia L et al. tor H related 5 (CFHR5) are associated with membranoproli- [Long-term effect of angiotensin-converting inhibitors in ferative glomerulonephritis type II (dense deposit disease). J children with proteinuria]. An Esp Pediatr 2001; 55: 219–224 Med Genet 2006; 43: 582–589 34. Butani L. Angiotensin blockade in children with chronic glo- 52. Iatropoulos P, Noris M, Mele C et al. Complement gene var- merulonephritis and heavy proteinuria. Pediatr Nephrol 2005; iants determine the risk of immunoglobulin-associated 20: 1651–1654 35. Vivarelli M, Pasini A, Emma F. Eculizumab for the treatment MPGN and C3 glomerulopathy and predict long-term renal of dense-deposit disease. N Engl J Med 2012; 366: 1163–1165 outcome. Mol Immunol 2016; 71: 131–142 Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018 12 | G. Sparta ` et al. 57. Nicolas C, Vuiblet V, Baudouin V et al. C3 nephritic factor 53. Bomback AS. Eculizumab in the treatment of membranopro- liferative glomerulonephritis. Nephron Clin Pract 2014; 128: associated with C3 glomerulopathy in children. Pediatr 270–276 Nephrol 2014; 29: 85–94 54. Inman M, Prater G, Fatima H et al. Eculizumab-induced 58. Haffner K, Michelfelder S, Pohl M. Successful therapy reversal of dialysis-dependent kidney failure from C3 glo- of C3Nef-positive C3 glomerulopathy with plasma therapy merulonephritis. Clin Kidney J 2015; 8: 445–448 and immunosuppression. Pediatr Nephrol 2015; 30: 1951–1959 55. Rodriguez-Osorio L, Ortiz A. Timing of eculizumab ther- 59. Rousset-Rouviere C, Cailliez M, Garaix F et al. Rituximab fails apy for C3 glomerulonephritis. Clin Kidney J 2015; 8: where eculizumab restores renal function in C3nef-related 449–452 DDD. Pediatr Nephrol 2014; 29: 1107–1111 56. Vivarelli M, Emma F. Treatment of C3 glomerulopathy with 60. Valenzuela NM, McNamara JT, Reed EF. Antibody-mediated graft complement blockers. Semin Thromb Hemost 2014; 40: injury: complement-dependent and complement-independent 472–477 mechanisms. Curr Opin Organ Transplant 2014; 19: 33–40 Downloaded from https://academic.oup.com/ckj/advance-article-abstract/doi/10.1093/ckj/sfy006/4915394 by Ed 'DeepDyve' Gillespie user on 12 July 2018
Clinical Kidney Journal – Oxford University Press
Published: Feb 28, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera