Evaluating the clinical utility of measuring levels of factor H and the related proteins.

After years of disappointing clinical results, the tide has finally changed and complement targeted-therapies have become a validated and accepted treatment option for several diseases. These accomplishments have revitalized the field and brought renewed attention to the prospects that complement therapeutics can offer. Streamlining diagnostics and therapeutics is imperative in this new era of clinical use of complement therapeutics. However, the incredible success in therapeutics has not been accompanied by the development of novel standardized tools for complement testing. Complement biomarkers can assist in the risk assessment and diagnosis of diseases as well as the prediction of disease progression and treatment response. Recently, a group of complement proteins has been suggested to be highly relevant in various complement-associated disorders, namely the human factor H (FH) protein family. This family of closely related proteins consists of FH, FH-like protein 1, and five factor H-related proteins, and they have been linked to eye, kidney, infectious, vascular, and autoimmune diseases as well as cancer. The goal of this review is to provide a comprehensive overview of the available data on circulating levels of FH and its related proteins in different pathologies. In addition, we examined the current literature to determine the clinical utility of measuring levels of the FH protein family in health and disease. Finally, we discuss future steps that are needed to make their clinical translation a reality.

How novel structures inform understanding of complement function.

During the last decade, the complement field has experienced outstanding advancements in the mechanistic understanding of how complement activators are recognized, what C3 activation means, how protein complexes like the C3 convertases and the membrane attack complex are assembled, and how positive and negative complement regulators perform their function. All of this has been made possible mostly because of the contributions of structural biology to the study of the complement components. The wealth of novel structural data has frequently provided support to previously held knowledge, but often has added alternative and unexpected insights into complement function. Here, we will review some of these findings focusing in the alternative and terminal complement pathways.

The development of atypical hemolytic uremic syndrome depends on complement C5.

Gene variants in the alternative pathway of the complement system strongly associate with atypical hemolytic uremic syndrome (aHUS), presumably by predisposing to increased complement activation within the kidney. Complement factor H (CFH) is the major regulator of complement activation through the alternative pathway. Factor H-deficient mice transgenically expressing a mutant CFH protein (Cfh(-/-).FHΔ16-20) that functionally mimics the CFH mutations reported in aHUS patients spontaneously develop thrombotic microangiopathy. To investigate the role of complement C5 activation in this aHUS model, we generated C5-deficient Cfh(-/-).FHΔ16-20 mice. Both C5-sufficient and C5-deficient Cfh(-/-).FHΔ16-20 mice had abnormal C3 deposition within the kidney, but spontaneous aHUS did not develop in any of the C5-deficient mice. Furthermore, although Cfh(-/-).FHΔ16-20 animals demonstrated marked hypersensitivity to experimentally triggered renal injury, animals with concomitant C5 deficiency did not. These data demonstrate a critical role for C5 activation in both spontaneous aHUS and experimentally triggered renal injury in animals with defective complement factor H function. This study provides a rationale to investigate therapeutic inhibition of C5 in human aHUS.

Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis.

BACKGROUND: Complement is a key component of the innate immune system, and variation in genes that regulate its activation is associated with renal and other disease. We aimed to establish the genetic basis for a familial disorder of complement regulation associated with persistent microscopic haematuria, recurrent macroscopic haematuria, glomerulonephritis, and progressive renal failure. METHODS: We sought patients from the West London Renal and Transplant Centre (London, UK) with unusual renal disease and affected family members as a method of identification of new genetic causes of kidney disease. Two families of Cypriot origin were identified in which renal disease was consistent with autosomal dominant transmission and renal biopsy of at least one individual showed C3 glomerulonephritis. A mutation was identified via a genome-wide linkage study and candidate gene analysis. A PCR-based diagnostic test was then developed and used to screen for the mutation in population-based samples and in individuals and families with renal disease. FINDINGS: Occurrence of familial renal disease cosegregated with the same mutation in the complement factor H-related protein 5 gene (CFHR5). In a cohort of 84 Cypriots with unexplained renal disease, four had mutation in CFHR5. Overall, we identified 26 individuals with the mutation and evidence of renal disease from 11 ostensibly unrelated kindreds, including the original two families. A mutant CFHR5 protein present in patient serum had reduced affinity for surface-bound complement. We term this renal disease CFHR5 nephropathy. INTERPRETATION: CFHR5 nephropathy accounts for a substantial burden of renal disease in patients of Cypriot origin and can be diagnosed with a specific molecular test. The high risk of progressive renal disease in carriers of the CFHR5 mutation implies that isolated microscopic haematuria or recurrent macroscopic haematuria should not be regarded as a benign finding in individuals of Cypriot descent. FUNDING: UK Medical Research Council and Wellcome Trust.

Treatment with human complement factor H rapidly reverses renal complement deposition in factor H-deficient mice.

Total deficiency of complement factor H (CFH) is associated with dense deposit disease and atypical hemolytic uremic syndrome. CFH is the major regulator of the alternative pathway of complement activation and its complete deficiency results in uncontrolled C3 activation through this pathway and secondary C3 deficiency. Plasma infusion, as a source of CFH, has been used with variable success to treat renal disease associated with its deficiency. However, the risks of volume and protein overload limit this therapeutic approach. In this study, we investigated the efficacy of a purified human CFH (hCFH) preparation in Cfh-gene knockout mice. These mice spontaneously develop both secondary plasma C3 deficiency and a renal abnormality characterized by massive accumulation of C3 along the glomerular basement membrane. The renal lesion is analogous to human dense deposit disease. Treatment of knockout mice with hCFH resulted in rapid normalization of plasma C3 levels and resolution of the glomerular basement membrane C3 deposition. Long-term treatment of mice with hCFH was not possible because of the development of an immune response against hCFH. Hence, we suggest that hCFH can be an effective alternative therapy to plasma infusions in patients with renal disease associated with CFH deficiency.

Complement factor H binds to denatured rather than to native pentameric C-reactive protein.

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.

Mutations in proteins of the alternative pathway of complement and the pathogenesis of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome is associated with mutations in the complement proteins factor H, factor I, factor B, C3, or membrane cofactor protein in about 50% of patients. The evolution and prognosis of the disease in patients carrying mutations in factor H is particularly poor, and renal transplantation most often fails because of recurrence of the disease in the graft. The risk of rapid loss of renal function in patients with functional mutations in factor H requires that effective treatment be initiated as soon as possible, but identification of these patients relies on genetic studies that are time consuming. We describe a case in which an in vitro hemolytic assay proved useful for rapidly assessing factor H dysfunction and for testing whether this dysfunction could be corrected with fresh frozen plasma. In the context of this case, we summarize recent advances in understanding the molecular mechanisms contributing to atypical hemolytic uremic syndrome, including descriptions of DNA- and protein-based analysis. We conclude that functional analysis of factor H should help rationalize the plasma treatment of patients with atypical hemolytic uremic syndrome.

Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains.

Factor H (FH) is an abundant serum glycoprotein that regulates the alternative pathway of complement-preventing uncontrolled plasma C3 activation and nonspecific damage to host tissues. Age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), and membranoproliferative glomerulonephritis type II (MPGN2) are associated with polymorphisms or mutations in the FH gene (Cfh), suggesting the existence of a genotype-phenotype relationship. Although AMD and MPGN2 share pathological similarities with the accumulation of complement-containing debris within the eye and kidney, respectively, aHUS is characterized by renal endothelial injury. This pathological distinction was reflected in our Cfh association analysis, which demonstrated that although AMD and MPGN2 share a Cfh at-risk haplotype, the haplotype for aHUS was unique. FH-deficient mice have uncontrolled plasma C3 activation and spontaneously develop MPGN2 but not aHUS. We show that these mice, transgenically expressing a mouse FH protein functionally equivalent to aHUS-associated human FH mutants, regulate C3 activation in plasma and spontaneously develop aHUS but not MPGN2. These animals represent the first model of aHUS and provide in vivo evidence that effective plasma C3 regulation and the defective control of complement activation on renal endothelium are the critical events in the molecular pathogenesis of FH-associated aHUS.