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Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh +/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh -/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function., Competing Interests: Declaration of Competing Interest Verena Aumiller and Steffen Schubert are employed by Silence Therapeutics GmbH. Marina Noris has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. Ariela Benigni and Giuseppe Remuzzi have consultancy agreements with BioCryst Pharmaceuticals., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Rational options for the treatment of end-stage renal disease (ESRD) due to atypical hemolytic uremic syndrome (aHUS) in children are still open to discussion. In the case of human complement factor H (CFH) deficiency, the choice is either kidney transplantation in combination with eculizumab, a humanized anti-C5 monoclonal antibody, or a combined liver-kidney transplantation. Case-Diagnosis/treatment: A child with a homozygous CFH deficiency underwent a successful liver-kidney transplantation. CFH levels normalized within days. After 6 years of follow-up, the graft function (Cockroft clearance 100 ml min 1.73 m) and the liver functions were normal. Results and Conclusions: The results of this long-term follow-up confirm that combined liver-kidney transplantation remains a reasonable option in patients with ESRD due to aHUS when an identified genetic abnormality of the C3 convertase regulator synthesized in the liver has been identified. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration is the leading cause of blindness in the elderly. The Y402H polymorphism in complement factor H promotes disease-like pathogenesis, and a Cfh +/- murine model can replicate this phenotype, but only after two years. We reasoned that by combining CFH deficiency with cigarette smoke exposure, we might be able to accelerate disease progression to facilitate preclinical research in this disease. Wild-type and Cfh +/- mice were exposed to nose-only cigarette smoke for three months. Retinal tissue morphology and visual function were evaluated by optical coherence tomography, fundus photography and autofluorescence, and electroretinogram. Retinal pigment epithelial cell phenotype and ultrastructure were evaluated by immunofluorescence staining and transmission electron microscopy. Cfh +/- smoking mice showed a dome-like protruding lesion at the ellipsoid zone (drusen-like deposition), many retinal hyper-autofluorescence spots, and a marked decrease in A- and B-wave amplitudes. Compared with non-smoking mice, wild-type and Cfh +/- smoking mice showed sub-retinal pigment epithelium complement protein 3 deposition, activation of microglia, metabolic waste accumulation, and impairment of tight junctions. Microglia cells migrated into the photoreceptor outer segment layer in Cfh +/- smoking mice showed increased activation. Our results suggest that exposing Cfh +/- mice to smoking leads to earlier onset of age-related macular degeneration than in other animal models, which may facilitate preclinical research into the pathophysiology and treatment of this disease.

C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH 1-5^18-20^R1-2 )], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH K D =505 nM; mFH K D =1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH -/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH -/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH -/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G., Competing Interests: KM has received research funding from Gemini Therapeutics, Idorsia Pharmaceuticals Ltd and Catalyst Biosciences as well as consultancy income from Freeline Therapeutics, Bath ASU & MPM Capital. MP provides consultancy for Alexion, Apellis, Gemini and Gyroscope Pharma. DK has received consultancy income from Gyroscope Therapeutics, Alexion Pharmaceuticals, Novartis, Apellis; Sarepta. TH is an employee of Gyroscope Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamala, Malik, Hallam, Cox, Yang, Vyas, Luli, Connelly, Gibson, Smith-Jackson, Denton, Pappworth, Huang, Kavanagh, Pickering and Marchbank.)

Deficiency of Complement Factor H Related (CFHR) plasma proteins and Autoantibody Positive Hemolytic Uremic Syndrome (DEAP-HUS) is a subtype of atypical hemolytic uremic syndrome, known to be associated with significant morbidity. Its pathogenesis is linked to the production of IgG autoantibodies against complement factor H, a regulator of the alternative complement pathway. The binding of the autoantibodies to the C terminal of complement factor H interferes with its regulatory function, leading to increased activation of the alternative complement pathway and consequent endothelial cellular damage. Early diagnosis and initiation of appropriate therapy is reported to lead to favorable outcomes. Institution of plasma exchange therapy within 24 h of diagnosis has been shown to rapidly lower antibody levels, leading to clinical improvement. Adjunctive immunosuppression therapy suppresses antibody production and helps in maintaining long-term clinical remission of the disease. Available data advocates a treatment regimen that combines plasma therapy (preferably plasma exchange) and immunosuppression to halt disease process and sustain long-term disease remission.

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen., Competing Interests: KM has received consultancy or research income from Gemini Therapeutics Inc, Freeline Therapeutics, MPM Capital, Idorsia Pharmaceuticals Ltd and Catalyst Biosciences. PB is a scientific co-founder of, and has received consultancy and research income from, Gemini Therapeutics Inc. AH is founder and Chief Scientific Officer of Invizius. EM is currently an employee of Invizius. AR has been employed at GlaxoSmithKline since October 2014. Results reported in this work were undertaken during her Wellcome Trust Intermediate Clinical Fellowship. Her spouse, David Kavanagh, is head of the National Renal Complement Therapeutics Centre, UK, and a board member and scientific advisor to Gyroscope Therapeutics Ltd. PNB and AR are co-authors on a recombinant CFH patent application (EP10803266A). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kerr, Herbert, Makou, Abramczyk, Malik, Lomax-Browne, Yang, Pappworth, Denton, Richards, Marchbank, Pickering and Barlow.)

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

Dengue disease is an inflammatory-driven pathology, and complement overactivation is linked to disease severity and vascular leakage. Additionally, dysregulation of complement alternative pathway (AP) components has been described, such as upregulation of complement factor D and downregulation of complement factor H (FH), which activate and inhibit the AP, respectively. Thus, the pathology of severe dengue could in part result from AP dysfunction, even though complement and AP activation usually provide protection against viral infections. In dengue virus-infected macrophages and endothelial cells (ECs), the site of replication and target for vascular pathology, respectively, the AP is activated. The AP activation, reduced FH and vascular leakage seen in dengue disease in part parallels other complement AP pathologies associated with FH deficiency, such as atypical haemolytic uraemic syndrome (aHUS). aHUS can be therapeutically targeted with inhibitors of complement terminal activity, raising the idea that strategies such as inhibition of complement or delivery of FH or other complement regulatory components to EC may be beneficial to combat the vascular leakage seen in severe dengue., (© 2020 Federation of European Biochemical Societies.)

Polymorphisms in the Complement Factor H (CFH) gene, coding for the Factor H protein (FH), can increase the risk for age-related macular degeneration (AMD). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. Whether this alone suffices to trigger AMD pathogenesis remains unclear. In AMD, retinal homeostasis is compromised due to the dysfunction of retinal pigment epithelium (RPE) cells. To investigate the impact of endogenous FH loss on RPE cell balance, we silenced CFH in human hTERT-RPE1 cells. FH reduction led to accumulation of C3, at both RNA and protein level and increased RPE vulnerability toward oxidative stress. Mild hydrogen-peroxide exposure in combination with CFH knock-down led to a reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation, which is a key aspect of AMD pathogenesis. In parallel, cell viability was decreased. The perturbations of energy metabolism were accompanied by transcriptional deregulation of several glucose metabolism genes as well as genes modulating mitochondrial stability. Our data suggest that endogenously produced FH contributes to transcriptional and metabolic homeostasis and protects RPE cells from oxidative stress, highlighting a novel role of FH in AMD pathogenesis.

Complement factor H (CFH) has a pivotal role in regulating alternative complement activation through its ability to inhibit the cleavage of the central complement component C3, which links innate and humoral immunity. However, insights into the role of CFH in B cell biology are limited. Here, we demonstrate that deficiency of CFH in mice leads to altered splenic B cell development characterized by the accumulation of marginal zone (MZ) B cells. Furthermore, B cells in Cf h -/- mice exhibit enhanced B cell receptor (BCR) signaling as evaluated by increased levels of phosphorylated Bruton's tyrosine kinase (pBTK) and phosphorylated spleen tyrosine kinase (pSYK). We show that enhanced BCR activation is associated with uncontrolled C3 consumption in the spleen and elevated complement receptor 2 (CR2, also known as CD21) levels on the surface of mature splenic B cells. Moreover, aged Cf h -/- mice developed splenomegaly with distorted spleen architecture and spontaneous B cell-dependent autoimmunity characterized by germinal center hyperactivity and a marked increase in anti-double stranded DNA (dsDNA) antibodies. Taken together, our data indicate that CFH, through its function as a complement repressor, acts as a negative regulator of BCR signaling and limits autoimmunity.

We report a case of an 18-month-old boy with H factor deficiency with atypical presentation: recurrent acute otitis media and several maternal family members with autoimmune disorders (vitiligo, thyroiditis and immune trombocytopenia). Blood tests revealed low C3 and AH50, as well as low properdin and H factor. I factor was normal. CFH gene molecular test confirmed the H factor deficiency diagnosis. This child had none of the typical manifestations of this disorder, namely Neisseria meningitidis infection or renal disease (glomerulonephritis and atypical haemolytic uremic syndrome). Autoimmune family history and correct interpretation of blood tests' results were crucial for this diagnosis.

Complement C3 glomerulopathy refers to a disease process in which abnormal control of complement activation or degradation results in predominant C3 fragment deposition within the glomerulus and causes glomerular damage. Abnormal control of the complement alternative pathway is a well-established risk factor for the occurrence of C3 glomerulonephritis. It is the first reported case in Iran with multiple mutations in complement factor H, with one of these mutations we have expected in hemolytic uremic syndrome rather than C3 glomerulopathy Genetic analysis showed that the molecular abnormalities of factor H led to complement factor H malfunction that were polymorphous and not restricted to the C-terminal domains of the protein.

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We herein reported a 4-month-old boy with transplantation-associated atypical hemolytic uremic syndrome (TA-aHUS) who was successfully treated with eculizumab. The patient diagnosed with type 3 of familial hemophagocytic lymphohistiocytosis underwent cord blood transplantation. After transplantation, he developed TA-aHUS, but plasma exchanges were unsuccessful. We identified deletions in CFH-related gene 1 (del-CFHR1) by the multiplex ligation-dependent probe amplification testing procedure and CFH autoantibodies. Eculizumab has been administered to the patient, with a marked improvement being achieved in thrombocytopenia. He has been well except for the persistent microhematuria for a year after transplantation. Uncontrolled complement activation might be involved in the pathophysiology of TA-aHUS.

C3 glomerulopathy is a potentially life-threatening disease of the kidney caused by dysregulated alternative pathway complement activation. The specific complement mediator(s) responsible for kidney injury in C3 glomerulopathy are yet to be defined and no specific therapy is currently available. We previously developed a mouse model of lethal C3 glomerulopathy with factor H and properdin gene double mutations. Therefore, we used this model to examine the role of C5 and C5a receptor (C5aR) in the pathogenesis of the disease. Disease severity in these factor H/properdin double-mutant mice was found to be correlated with plasma C5 levels, and prophylactic anti-C5 mAb therapy was effective in preventing lethal C3 glomerulopathy. When given to these double-mutant mice that had already developed active disease with severe proteinuria, anti-C5 mAb treatment also prevented death in half of the mice. Deficiency of C5aR significantly reduced disease severity, suggesting that C5aR-mediated inflammation contributed to C3 glomerulopathy. Thus, C5 and C5aR have a critical role in C3 glomerulopathy. Hence, early intervention targeting these pathways may be an effective therapeutic strategy for patients with C3 glomerulopathy., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

To evaluate the association among complement factor H-related ( CFHRs ) gene deficiency, complement factor H (CFH) autoantibodies, and atypical hemolytic uremic syndrome (aHUS) susceptibility. EMBASE, PubMed, and the ISI Web of Science databases were searched for all eligible studies on the relationship among CFHRs deficiency, anti-FH autoantibodies, and aHUS risk. Eight case-control studies with 927 cases and 1182 controls were included in this study. CFHR1 deficiency was significantly associated with an increased risk of aHUS (odds ratio (OR) = 3.61, 95% confidence interval (95% CI), 1.96, 6.63, p < 0.001), while no association was demonstrated in individuals with only CFHR1/R3 deficiency (OR = 1.32, 95% CI, 0.50, 3.50, p = 0.56). Moreover, a more significant correlation was observed in people with both FH-anti autoantibodies and CFHR1 deficiency (OR = 11.75, 95% CI, 4.53, 30.44, p < 0.001) in contrast to those with only CFHR1 deficiency. In addition, the results were essentially consistent among subgroups stratified by study quality, ethnicity, and gene detection methods. The present meta-analysis indicated that CFHR1 deletion was significantly associated with the risk of aHUS, particularly when combined with anti-FH autoantibodies, indicating that potential interactions among CFHR1 deficiency and anti-FH autoantibodies might impact the risk of aHUS., Competing Interests: None of the authors have any conflicts of interest to disclose. All authors had access to all the study data, take responsibility for the accuracy of the analysis, and had authority over manuscript preparation and the decision to submit the manuscript for publication. All authors approve the manuscript and agree to adhere to all terms outlined in Annals of Internal Medicine information for authors including terms for copyright.

Activation of complement generates C5a which leads to signaling through C5aR1. This is tightly controlled, including by the plasma proteins factor H (FH) and carboxypeptidase N. Here we studied a chronic serum sickness (CSS) model of glomerulonephritis (GN) in which there is an active humoral immune response, formation of glomerular immune complexes (ICs), and resulting glomerular inflammation. The antibody response, glomerular IC deposition, the degree of GN, and consequent renal functional insufficiency in CSS were all worse in FH-/- mice compared to wild-type FH+/+ animals. This was ameliorated in the former by giving a C5aR1 antagonist for the final 3 weeks of the 5-week protocol. In contrast, blocking CP-mediated inactivation of C5a increased these disease measures. Thus, complement regulation by both plasma FH and CP to limit the quantity of active C5a is important in conditions where the humoral immune response is directed to a continuously present foreign antigen. Signaling through C5aR1 enhances the humoral immune response as well as the inflammatory response to ICs that have formed in glomeruli. Both effects are relevant even after disease has begun. Thus, pharmacological targeting of C5a in IC-mediated GN has potential clinical relevance.

Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh(-/-)) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh(-/-) +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis.

Extra-cellular deposition including amyloid beta (Aβ) is a feature of retinal ageing. It has been documented for Bruch's membrane (BM) where Aβ is elevated in complement factor H knockout mice (Cfh(-/-)) proposed as a model for age related macular degeneration. However, arterial deposition in choroidal vessels prior to perfusion across BM has not been examined. Aβ is associated with tau phosphorylation and these are linked in blood vessels in Alzheimers Disease where they can drive perivascular pathology. Here we ask if Aβ, tau and phosphorylated tau are features of ageing in choroidal vessels in 12 month C57 BL/6 and Cfh(-/-) mice, using immune staining and Western blot analysis. Greater levels of Aβ and phosphorylated tau are found in choroidal vessels in Cfh(-/-) mice. Western blot revealed a 40% increase in Aβ in Cfh(-/-) over C57 BL/6 mice. Aβ deposits coat around 55% of the luminal wall in Cfh(-/-) compared to only about 40% in C57 BL/6. Total tau was similar in both groups, but phosphorylated tau increased by >100% in Cfh(-/-) compared to C57 BL/6 and covered >75% of the luminal wall compared to 50% in C57 BL/6. Hence, phosphorylated tau is a marked choroidal feature in this mouse model. Aβ deposition was clumped in Cfh(-/-) mice and likely to influence blood flow dynamics. Disturbed flow is associated with atherogenesis and may be related to the accumulation of membrane attack complex recently identified between choroidal vessels in those at high risk of macular degeneration due to complement factor H polymorphisms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The complement-mediated renal diseases C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) strongly associate with inherited and acquired abnormalities in the regulation of the complement alternative pathway (AP). The major negative regulator of the AP is the plasma protein complement factor H (FH). Abnormalities in FH result in uncontrolled activation of C3 through the AP and associate with susceptibility to both C3G and aHUS. Although previously developed FH-deficient animal models have provided important insights into the mechanisms underlying susceptibility to these unique phenotypes, these models do not entirely reproduce the clinical observations. FH is predominantly synthesized in the liver. We generated mice with hepatocyte-specific FH deficiency and showed that these animals have reduced plasma FH levels with secondary reduction in plasma C3. Unlike mice with complete FH deficiency, hepatocyte-specific FH-deficient animals developed neither plasma C5 depletion nor accumulation of C3 along the glomerular basement membrane. In contrast, subtotal FH deficiency associated with mesangial C3 accumulation consistent with C3G. Although there was no evidence of spontaneous thrombotic microangiopathy, the hepatocyte-specific FH-deficient animals developed severe C5-dependent thrombotic microangiopathy after induction of complement activation within the kidney by accelerated serum nephrotoxic nephritis. Taken together, our data indicate that subtotal FH deficiency can give rise to either spontaneous C3G or aHUS after a complement-activating trigger within the kidney and that the latter is C5 dependent., (Copyright © 2016 by the American Society of Nephrology.)

Factor H (FH) binds apoptotic cells to limit the inflammatory potential of complement. Here we report that FH is actively internalized by apoptotic cells to enhance cathepsin L-mediated cleavage of endogenously expressed C3, which results in increased surface opsonization with iC3b. In addition, internalized FH forms complexes with nucleosomes, facilitates their phagocytosis by monocytes and induces an anti-inflammatory biased cytokine profile. A similar cytokine response was noted for apoptotic cells coated with FH, confirming that FH diminishes the immunogenic and inflammatory potential of autoantigens. These findings were supported by in vivo observations from CFH(-/-) MRL-lpr mice, which exhibited higher levels of circulating nucleosomes and necrotic cells than their CFH(+/+) littermates. This unconventional function of FH broadens the established view of apoptotic cell clearance and appears particularly important considering the strong associations with genetic FH alterations and diseases such as systemic lupus erythematosus and age-related macular degeneration.

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh(-/-)), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh(-/-) mice. This effect was found to be dependent on CR3 expression on bone marrow-derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b-CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Kidney diseases caused by genetic or acquired dysregulation of the complement alternative pathway (AP) are traditionally classified on the basis of clinical presentation (atypical hemolytic uremic syndrome as thrombotic microangiopathy), biopsy appearance (dense deposit disease and C3 GN), or clinical course (atypical postinfectious GN). Each is characterized by an inappropriate activation of the AP, eventuating in renal damage. The clinical diversity of these disorders highlights important differences in the triggers, the sites and intensity of involvement, and the outcome of the AP dysregulation. Nevertheless, we contend that these diseases should be grouped as disorders of the AP and classified on an etiologic basis. In this review, we define different pathophysiologic categories of AP dysfunction. The precise identification of the underlying abnormality is the key to predict the response to immune suppression, plasma infusion, and complement-inhibitory drugs and the outcome after transplantation. In a patient with presumed dysregulation of the AP, the collaboration of the clinician, the renal pathologist, and the biochemical and genetic laboratory is very much encouraged, because this enables the elucidation of both the underlying pathogenesis and the optimal therapeutic approach., (Copyright © 2015 by the American Society of Nephrology.)

Although glomerular immunoglobulin M (IgM) deposition occurs in a variety of glomerular diseases, the mechanism of deposition and its clinical significance remain controversial. Some have theorized IgM becomes passively trapped in areas of glomerulosclerosis. However, recent studies found that IgM specifically binds damaged glomeruli. Therefore, we tested whether natural IgM binds to neo-epitopes exposed after insults to the glomerulus and exacerbates disease in mice deficient in the complement regulatory protein factor H; a model of non-sclerotic and nonimmune-complex glomerular disease. Immunofluorescence microscopy demonstrated mesangial and capillary loop deposition of IgM, whereas ultrastructural analysis found IgM deposition on endothelial cells and subendothelial areas. Factor H-deficient mice lacking B cells were protected from renal damage, as evidenced by milder histologic lesions on light and electron microscopy. IgM, but not IgG, from wild-type mice bound to cultured murine mesangial cells. Furthermore, injection of purified IgM into mice lacking B cells bound within the glomeruli and induced proteinuria. A monoclonal natural IgM-recognizing phospholipids also bound to glomeruli in vivo and induced albuminuria. Thus, our results indicate specific IgM antibodies bind to glomerular epitopes and that IgM contributes to the progression of glomerular damage in this mouse model of non-sclerotic glomerular disease.

Background: Neonatal atypical hemolytic uremic syndrome (aHUS) is a rare but severe disease that is mainly due to methylmalonic aciduria or genetic complement abnormalities. Traditional management of aHUS includes plasma infusion/exchange, but in small or unstable infants, plasma exchange can be challenging because of high extracorporeal volume and difficulty to obtain an adequate venous access. The C5 complement blocker eculizumab has become a cornerstone of first-line management of aHUS due to complement deregulation in older patients. However, little data are available on its use in neonatal aHUS., Case-Diagnosis/treatment: We report on an 11-day-old neonate with severe aHUS (myocardial impairment, respiratory failure, acute kidney disease requiring hemodiafiltration) due to homozygous factor-H deficiency. She received early treatment with eculizumab as first-line therapy and completely recovered within 5 days. A second dose of eculizumab was administered 7 days after the first infusion, followed by a dose every 2 weeks for 2 months and then every 3 weeks, at the same dosage (300 mg). With more than 24 months of follow-up, renal function remains normal., Conclusions: We report on the long-term efficacy and safety of eculizumab as first-line therapy in neonatal aHUS. However its use still requires optimization in terms of indications and administration (frequency, dosage).

Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN., (Copyright © 2014 by the American Society of Nephrology.)

Background: Dense deposit disease (DDD) (also known as membranoproliferative glomerulonephritis type II) in childhood is a rare glomerulonephritis with frequent progression to end-stage renal disease (ESRD) and a high recurrence after kidney transplantation. The pathophysiologic basis of DDD is associated with the uncontrolled systemic activation of the alternative pathway (AP) of the complement cascade., Case-Diagnosis/treatment: A 14-year-old girl presented with edema and nephrotic range proteinuria. Blood tests showed hypoalbuminemia, nephrotic range proteinuria, normal renal function, and a low C3 level. Renal biopsy confirmed the diagnosis of crescentic DDD. Complement analysis revealed strong AP activation (low C3), positive C3 nephritic factor (C3NeF), and a decreased complement factor H (CFH) levels with CFH polymorphisms. Therapy with eculizumab was considered after the failure of corticosteroid and plasmapheresis to modulate the ongoing massive proteinuria and persistence of low serum C3 levels. There was a marked clinical and biochemical response following the administration of eculizumab., Conclusions: Our case emphasizes the efficacy of eculizumab in the management of crescentic DDD in a patient with a normal renal function, in a short follow-up period. Considering previously reported cases, it appears that eculizumab represents a promising new approach which may prevent progression to ESRD in a subset of patients with DDD.

Background: Deficiency of complement factor H-related (CFHR) proteins and CFH autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) represents a unique subgroup of complement-mediated atypical HUS (aHUS). Autoantibodies to the C-terminus of CFH block CFH surface recognition and mimic mutations found in the genetic form of (CFH-mediated) aHUS. CFH autoantibodies are found in 10-15 % of aHUS patients and occur--so far unexplained--almost exclusively in the background of CFHR1 or CFHR3/CFHR1 deletions., Methods: As a well-defined role for eculizumab in the treatment of complement-mediated aHUS is becoming established, its role in DEAP-HUS is less conspicuous, where a B-cell-depleting and immunosuppressive treatment strategy is being proposed in the literature., Results: We here show eculizumab to be safe and effective in maintaining a disease-free state, without recurrence, in a previously plasma-therapy-dependent DEAP-HUS patient, and in another patient in whom, although showing a good clinical response to plasma therapy, the therapy was hampered by allergic reactions to fresh frozen plasma and contend there is a rationale for the use of eculizumab in concert with an immunosuppressive strategy in the treatment of DEAP-HUS. Considering the high rate of early relapse, the possible coexistence and contribution of both known and unknown complement-gene mutations, the probable pathogenic role of CFHR1 as a complement alternative pathway (CAP) regulator, the experimental nature of measuring and using anti-CFH autoantibodies to guide management, and until the positive reports of immunosuppression in addition to plasma therapy are confirmed in prospective studies, we feel that a complement-directed therapy should not be neglected in DEAP-HUS. Serial CFH autoantibody titer testing may become a valuable tool to monitor treatment response, and weaning patients off eculizumab may become an option once CFH autoantibody levels are depleted., Conclusions: A prospective study of eculizumab treatment in a larger cohort of DEAP-HUS patients is required to validate the applicability of our positive experience.

Uncontrolled activation of the complement alternative pathway is associated with complement-mediated renal disease. Factor B and factor D are essential components of this pathway, while factor H (FH) is its major regulator. In complete FH deficiency, uncontrolled C3 activation through the alternative pathway results in plasma C3 depletion and complement-mediated renal disease. These are dependent on factor B. Mannan-binding lectin-associated serine proteases 1 and 3 (MASP-1, MASP-3) have been shown recently to contribute to alternative pathway activation by cleaving pro-factor D to its active form, factor D. We studied the contribution of MASP-1 and MASP-3 to uncontrolled alternative pathway activation in experimental complete FH deficiency. Co-deficiency of FH and MASP-1/MASP-3 did not ameliorate either the plasma C3 activation or glomerular C3 accumulation in FH-deficient mice. Our data indicate that MASP-1 and MASP-3 are not essential for alternative pathway activation in complete FH deficiency., (© 2013 The Authors. Clinical and Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Atypical hemolytic uremic syndrome (aHUS) evolves into end-stage renal failure in nearly half of affected patients and is associated with defective regulation of the alternative complement pathway. Patients with a complement factor H (CFH) mutation have a 30-100% risk of graft loss due to aHUS recurrence or graft thrombosis. Since CFH is produced predominantly by the liver, combined liver-kidney transplant is a curative treatment option. One major unexpected risk includes liver failure secondary to uncontrolled complement activation. We report a successful combined liver-kidney transplantation with perioperative plasma exchange and use of the humanized anti-C5 monoclonal antibody eculizumab., Case Diagnosis/treatment: An 11-month-old female presented with oliguric renal failure after 3 weeks of flu-like symptoms in the absence of diarrhea. Following the identification of Escherichia coli 0157:H7 in her stool, she was discharged home on peritoneal dialysis with a diagnosis of Shiga toxin-associated HUS. Three months later, she developed severe anemia, thrombocytopenia, and neurological involvement. aHUS was diagnosed and confirmed, and genetic testing revealed a mutation in CFH SCR20. Once donor organs became available, she received preoperative plasma exchange followed by eculizumab infusion with intra-operative fresh frozen plasma prior to combined liver-kidney transplant. At 19 months post-transplant, she continues to have excellent allograft and liver function without signs of disease recurrence., Conclusion: Perioperative use of eculizumab in conjunction with plasma exchange during simultaneous liver-kidney transplant can be used to inhibit terminal complement activity, thereby optimizing successful transplantation by reducing the risk of graft thrombosis.

Variations in several complement genes are now known to be significant risk factors for the development of age-related macular degeneration (AMD). Despite dramatic effects on disease susceptibility, the underlying mechanisms by which common polymorphisms in complement proteins alter disease risk have remained unclear. Genetically modified mice in which the activity of the complement has been altered are available and can be used to investigate the role of complement in the pathogenesis of AMD. In this mini review, we will discuss some existing complement models of AMD and our efforts to develop and characterize the ocular phenotype in a variety of mice in which complement is either chronically activated or inhibited. A spectrum of complement dysregulation was modeled on the APOE4 AMD mouse model by crossing these mice to complement factor H knockout (cfh-/-) mice to test the impact of excess complement activation, and by crossing them to soluble-complement-receptor-1-related protein y (sCrry) mice, in which sCrry acts as a potent inhibitor of mouse complement acting in a manner similar to CFH. In addition, we have also generated humanized CFH mice expressing normal and risk variants of CFH.

We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) deficiency associated with a homozygous CFH mutation (c.2880delT [p.Phe960fs]). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype (CD46GGAAC) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype (CD46AAGGT) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H-like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

C3 glomerulopathy refers to those renal lesions characterized histologically by predominant C3 accumulation within the glomerulus, and pathogenetically by aberrant regulation of the alternative pathway of complement. Dense deposit disease is distinguished from other forms of C3 glomerulopathy by its characteristic appearance on electron microscopy. The extent to which dense deposit disease also differs from other forms of C3 glomerulopathy in terms of clinical features, natural history, and outcomes of treatment including renal transplantation is less clear. We discuss the pathophysiology of C3 glomerulopathy, with evidence for alternative pathway dysregulation obtained from affected individuals and complement factor H (Cfh)-deficient animal models. Recent linkage studies in familial C3 glomerulopathy have shown genomic rearrangements in the Cfh-related genes, for which the novel pathophysiologic concept of Cfh deregulation has been proposed., (© 2013 Elsevier Inc. All rights reserved.)

Background: Renal insufficiency is increasingly common in end-stage liver disease and allocation of livers to this category of patient has escalated. The frequency of combined liver-kidney transplantation (CLKT) has consequently increased. Indications for CLKT in children differ from those for adults and typically include rare congenital conditions; subsequently limited numbers of this procedure have been performed in paediatric patients worldwide. Scant literature exists on the subject., Methods: Subsequent to institutional approval, a retrospective chart analysis of all paediatric CLKTs performed at the Transplant Unit, Wits Donald Gordon Medical Centre, University of the Witwatersrand, Johannesburg, South Africa between January 2005 and July 2013 was conducted., Results: Defining children as younger than 18 years of age, 43 patients had received a liver transplant since 2005, of whom 8 received a CLKT. Indications included autosomal recessive polycystic kidney disease (n=3), primary hyperoxaluria type 1 (n=4) and heterozygous factor H deficiency with atypical haemolytic uraemic syndrome (n=1). Graft combinations included whole liver and one kidney (n=5), whole liver and two kidneys (n=1) and left lateral liver segment and one kidney (n=2), all from deceased donors. Patient age ranged from 4 to 17 years (median 9) and included 4 females and 4 males. Weight ranged from 13 to 42 kg (median 22.5). We describe one in-hospital mortality. The remaining 7 patients were long-term survivors with a survival range from 6 to 65 months., Conclusions: Although rarely indicated in children, CLKT is an effective treatment option, appropriately utilising a scarce resource and significantly improving quality of life in the recipient.

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid β (Aβ) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aβ on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh(-/-) mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh(-/-) eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh(-/-) animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh(-/-) RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis.

Complement factor H (Cfh) is a key regulator of the complement cascade and protects C57BL/6 mice from immune complex-mediated complement-dependent glomerulonephritis. In chronic serum sickness (CSS) there are increased deposits of immune complexes in the glomeruli with inflammation and a scarring phenotype. As cucurmin is an effective anti-inflammatory agent and reduces complement activation, we hypothesized that it should alleviate renal disease in this setting. To determine the effectiveness of curcumin, an apoferritin-induced CSS model in Cfh-deficient (Cfh(-/-)) mice was used. Curcumin treatment (30 mg/kg) given every day in parallel with apoferritin reduced glomerulonephritis and enhanced kidney function (blood urea nitrogen, 45·4 ± 7·5 versus 35·6 ± 5·1; albuminuria, 50·1 ± 7·1 versus 15·7 ± 7·1; glomerulonephritis, 2·62 + 0·25 versus 2 + 0·3, P < 0·05). In line with reduced IgG deposits in mice with CSS given curcumin, C9 deposits were reduced indicating reduced complement activation. Mice treated with curcumin had a significant reduction in the number of splenic CD19(+) B cells and the ratio of CD19 : CD3 cells (P < 0·05) with no change in the T-cell population. Myeloperoxidase assay showed reduced macrophages in the kidney. However, a significant reduction in the M2 subset of splenic macrophages by apoferritin was prevented by curcumin, suggesting a protective function. Curcumin treatment reduced mRNA expression of inflammatory proteins monocyte chemoattractant protein-1 and transforming growth factor-β and matrix proteins, fibronectin, laminin and collagen. Our results clearly illustrate that curcumin reduces glomerulosclerosis, improves kidney function and could serve as a therapeutic agent during serum sickness., (© 2013 John Wiley & Sons Ltd.)