scholarly journals Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy

2020 ◽  
Vol 32 (1) ◽  
pp. 99-114
Author(s):  
Kishor Devalaraja-Narashimha ◽  
Karoline Meagher ◽  
Yifan Luo ◽  
Cong Huang ◽  
Theodore Kaplan ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Gene Mutations ◽  
Factor H ◽  
Complement Factor ◽  
C3 Glomerulopathy ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Pathologic Features ◽  
Complement Gene ◽  
Extended Survival

BackgroundC3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches.MethodsA novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice.ResultsThe C3hu/hu mice exhibit increased morbidity early in life and die by about 5–6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function.ConclusionsThe C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.

scholarly journals Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy

2020 ◽  
Vol 31 (2) ◽  
pp. 365-373 ◽  
Author(s):  
Adam P. Levine ◽  
Melanie M.Y. Chan ◽  
Omid Sadeghi-Alavijeh ◽  
Edwin K.S. Wong ◽  
H. Terence Cook ◽  
...  
Keyword(s):  
Large Scale ◽  
Rare Variants ◽  
Alternative Pathway ◽  
Atypical Hemolytic Uremic Syndrome ◽  
Gene Mutations ◽  
Whole Genome Sequence ◽  
European Ancestry ◽  
Whole Genome ◽  
C3 Glomerulopathy ◽  
Complement Gene

BackgroundPrimary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN.MethodsWe analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource–Rare Diseases Study. We examined copy number, rare, and common variants.ResultsOur analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (P=3.29×10−8; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (P=1.21×10−8; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure.ConclusionsWe found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.

scholarly journals An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy

2018 ◽  
Vol 29 (6) ◽  
pp. 1649-1661 ◽  
Author(s):  
Yi Yang ◽  
Harriet Denton ◽  
Owen R. Davies ◽  
Kate Smith-Jackson ◽  
Heather Kerr ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Alternative Pathway ◽  
Treatment Options ◽  
Chinese Hamster ◽  
Factor H ◽  
Complement Factor H ◽  
Complement Factor ◽  
C3 Glomerulopathy

Background C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1–5 linked to repeats 18–20 (FH1–5^18–20), that was effective in experimental C3G. However, the serum t1/2 of FH1–5^18–20 was significantly shorter than that of serum-purified FH.Methods We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1–2) at the carboxy or amino terminus of human FH1–5^18–20 to generate two homodimeric mini-FH constructs (FHR1–2^1–5^18–20 and FH1–5^18–20^R1–2, respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient Cfh−/− mice.Results FHR1–2^1–5^18–20 and FH1–5^18–20^R1–2 homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FHR1–2^1–5^18–20. Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity in vitro and restricted glomerular basement membrane C3 deposition in vivo significantly better than FH or FH1–5^18–20. FH1–5^18–20^R1–2 had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum t1/2 compared with that of FH1–5^18–20, and significantly better retention in the kidney than FH or FH1–5^18–20.Conclusions FH1–5^18–20^R1–2 may have utility as a treatment option for C3G or other complement-mediated diseases.

scholarly journals CFHR5 Genetic Variations and Serum Levels in Patients with Immune-Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

2020 ◽  
Author(s):  
Nóra Garam ◽  
Marcell Cserhalmi ◽  
Zoltán Prohászka ◽  
Ágnes Szilágyi ◽  
Nóra Veszeli ◽  
...  
Keyword(s):  
Immune Complex ◽  
Membranoproliferative Glomerulonephritis ◽  
Kidney Diseases ◽  
Alternative Pathway ◽  
Serum Levels ◽  
Genetic Alterations ◽  
Factor H ◽  
Complement Factor ◽  
C3 Glomerulopathy

Abstract Background: Factor H-related-5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune-complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement and follow-up data.Results: 120 patients with a histologically-proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger-sequencing, and selected mutants were studied as recombinant proteins in ELISA and SPR.Eight relevant CFHR5 variations in 14 patients (11.7%) were observed, 5 of them identified as pathogenic for C3G. The FHR-5G278S and FHR-5R356H mutations altered the interaction of FHR-5 with C3b, when compared to the FHR-5WT. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period, furthermore, it showed clear association with signs of hypocomplementemia and clinically meaningful clusters.Conclusions: Our observations support the hypothesis that FHR-5 protein and its genetic alterations play a role in the pathogenesis of IC-MPGN/C3G.

scholarly journals FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

2021 ◽  
Vol 12 ◽  
Author(s):  
Nóra Garam ◽  
Marcell Cserhalmi ◽  
Zoltán Prohászka ◽  
Ágnes Szilágyi ◽  
Nóra Veszeli ◽  
...  
Keyword(s):  
Immune Complex ◽  
Membranoproliferative Glomerulonephritis ◽  
Kidney Diseases ◽  
Alternative Pathway ◽  
Serum Levels ◽  
Factor H ◽  
Fine Tuning ◽  
Complement Factor ◽  
C3 Glomerulopathy

BackgroundFactor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data.MethodsA total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR).ResultsEight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters.ConclusionsOur observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G.

scholarly journals COVID-19: a trigger for severe thrombotic microangiopathy in a patient with complement gene variant

2022 ◽  
Vol 0 (0) ◽  
Author(s):  
Larisa Pinte ◽  
Bogdan Marian Sorohan ◽  
Zoltán Prohászka ◽  
Mihaela Gherghiceanu ◽  
Cristian Băicuş
Keyword(s):  
Hemolytic Uremic Syndrome ◽  
Thrombotic Microangiopathy ◽  
Alternative Pathway ◽  
Atypical Hemolytic Uremic Syndrome ◽  
Factor H ◽  
Complement C3 ◽  
Uncontrolled Hypertension ◽  
Complement Factor ◽  
Uremic Syndrome ◽  
Complement Gene

Abstract The evidence regarding thrombotic microangiopathy (TMA) related to Coronavirus Infectious Disease 2019 (COVID-19) in patients with complement gene mutations as a cause of acute kidney injury (AKI) are limited. We presented a case of a 23-year-old male patient admitted with an asymptomatic form of COVID-19, but with uncontrolled hypertension and AKI. Kidney biopsy showed severe lesions of TMA. In evolution patient had persistent microangiopathic hemolytic anemia, decreased level of haptoglobin and increased LDH level. Decreased complement C3 level and the presence of schistocytes were found for the first time after biopsy. Kidney function progressively decreased and the patient remained hemodialysis dependent. Complement work-up showed a heterozygous variant with unknown significance in complement factor I (CFI) c.-13G>A, affecting the 5' UTR region of the gene. In addition, the patient was found to be heterozygous for the complement factor H (CFH) H3 haplotype (involving the rare alleles of c.-331C>T, Q672Q and E936D polymorphisms) reported as a risk factor of atypical hemolytic uremic syndrome. This case of AKI associated with severe TMA and secondary hemolytic uremic syndrome highlights the importance of genetic risk modifiers in the alternative pathway dysregulation of the complement in the setting of COVID-19, even in asymptomatic forms.

scholarly journals CFH and CFHR Copy Number Variations in C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis

2021 ◽  
Vol 12 ◽  
Author(s):  
Rossella Piras ◽  
Matteo Breno ◽  
Elisabetta Valoti ◽  
Marta Alberti ◽  
Paraskevas Iatropoulos ◽  
...  
Keyword(s):  
Immune Complex ◽  
Single Molecule ◽  
Copy Number ◽  
Membranoproliferative Glomerulonephritis ◽  
Alternative Pathway ◽  
Copy Number Variations ◽  
Factor H ◽  
Complement C3 ◽  
Complement Factor ◽  
C3 Glomerulopathy

C3 Glomerulopathy (C3G) and Immune Complex-Mediated Membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases characterized by glomerular deposition of C3 caused by dysregulation of the alternative pathway (AP) of complement. In approximately 20% of affected patients, dysregulation is driven by pathogenic variants in the two components of the AP C3 convertase, complement C3 (C3) and Factor B (CFB), or in complement Factor H (CFH) and Factor I (CFI), two genes that encode complement regulators. Copy number variations (CNVs) involving the CFH-related genes (CFHRs) that give rise to hybrid FHR proteins also have been described in a few C3G patients but not in IC-MPGN patients. In this study, we used multiplex ligation-dependent probe amplification (MLPA) to study the genomic architecture of the CFH-CFHR region and characterize CNVs in a large cohort of patients with C3G (n = 103) and IC-MPGN (n = 96) compared to healthy controls (n = 100). We identified new/rare CNVs resulting in structural variants (SVs) in 5 C3G and 2 IC-MPGN patients. Using long-read single molecule real-time sequencing (SMRT), we detected the breakpoints of three SVs. The identified SVs included: 1) a deletion of the entire CFH in one patient with IC-MPGN; 2) an increased number of CFHR4 copies in one IC-MPGN and three C3G patients; 3) a deletion from CFHR3-intron 3 to CFHR3-3′UTR (CFHR34–6Δ) that results in a FHR3-FHR1 hybrid protein in a C3G patient; and 4) a CFHR31–5-CFHR410 hybrid gene in a C3G patient. This work highlights the contribution of CFH-CFHR CNVs to the pathogenesis of both C3G and IC-MPGN.

Molecular basis of complement-mediated renal disease

2015 ◽  
Author(s):  
Nicholas Medjeral-Thomas ◽  
Anna Richards ◽  
Matthew C. Pickering
Keyword(s):  
Haemolytic Uraemic Syndrome ◽  
Alternative Pathway ◽  
Protein Family ◽  
Factor H ◽  
Complement Factor ◽  
C3 Glomerulopathy ◽  
Ongoing Research ◽  
Dense Deposit ◽  
Atypical Haemolytic Uraemic Syndrome ◽  
Glomerular Lesions

Abnormal regulation of complement is intimately associated with C3 glomerulopathy and atypical haemolytic uraemic syndrome. Atypical haemolytic uraemic syndrome is characterized by renal thrombotic microangiopathy due to an inability to regulate complement activation along the renal endothelium. The development of thrombosis is critically dependent on the ability to activate C5. Eculizumab, a monoclonal anti-C5 antibody, is an effective therapy for this condition. C3 glomerulopathy refers to glomerular lesions characterized by accumulation of C3 in the absence of immunoglobulin. The prototypic example is dense deposit disease. This condition is associated with impaired regulation of the alternative pathway in plasma. In other subtypes of C3 glomerulopathy, familial studies have identified mutations within the complement factor H-related protein family. Polymorphic variation within this protein family also influences susceptibility to IgA nephropathy. The mechanism underlying these associations remains unknown and is the subject of ongoing research efforts.

scholarly journals New functional and structural insights from updated mutational databases for complement factor H, Factor I, membrane cofactor protein and C3

2014 ◽  
Vol 34 (5) ◽  
Author(s):  
Elizabeth Rodriguez ◽  
Pavithra M. Rallapalli ◽  
Amy J. Osborne ◽  
Stephen J. Perkins
Keyword(s):  
Alternative Pathway ◽  
Factor H ◽  
Complement Factor H ◽  
Complement C3 ◽  
Complement Factor ◽  
Membrane Cofactor Protein ◽  
Complement Alternative Pathway ◽  
Factor I ◽  
Mutational Hotspots ◽  
Structural Insights

A new compilation of 324 mutations in four major proteins from the complement alternative pathway reveals mutational hotspots in factor H and complement C3, and less so in factor I and membrane cofactor protein. Their associations with function are discussed.

scholarly journals De Novo Atypical Haemolytic Uremic Syndrome after Kidney Transplantation

2018 ◽  
Vol 2018 ◽  
pp. 1-4
Author(s):  
Arnaud Devresse ◽  
Martine de Meyer ◽  
Selda Aydin ◽  
Karin Dahan ◽  
Nada Kanaan
Keyword(s):  
Kidney Transplantation ◽  
De Novo ◽  
Alternative Pathway ◽  
Factor H ◽  
Complement Factor ◽  
Haemolytic Uremic Syndrome ◽  
Hinman Syndrome ◽  
Uremic Syndrome ◽  
Stage Renal Disease ◽  
End Stage

De novo thrombotic microangiopathy (TMA) can occur after kidney transplantation. An abnormality of the alternative pathway of complement must be suspected and searched for, even in presence of a secondary cause. We report the case of a 23-year-old female patient who was transplanted with a kidney from her mother for end-stage renal disease secondary to Hinman syndrome. Early after transplantation, she presented with 2 episodes of severe pyelonephritis, associated with acute kidney dysfunction and biological and histological features of TMA. Investigations of the alternative pathway of the complement system revealed atypical haemolytic uremic syndrome secondary to complement factor I mutation, associated with mutations in CD46 and complement factor H related protein genes. Plasma exchanges followed by eculizumab injections allowed improvement of kidney function without, however, normalization of creatinine.

scholarly journals A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

2021 ◽  
Vol 11 ◽  
Author(s):  
Orla M. Dunne ◽  
Xin Gao ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Penelope J. Adamson ◽  
...  
Keyword(s):  
Host Cell ◽  
Analytical Ultracentrifugation ◽  
Dissociation Constants ◽  
Alternative Pathway ◽  
Factor H ◽  
Regulatory Control ◽  
Size Exclusion ◽  
Complement Factor ◽  
Dimer Formation ◽  
Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

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