Inherited Kidney Complement Diseases.

In the past 20 years, we have witnessed tremendous advances in our ability to diagnose and treat genetic diseases of the kidney caused by complement dysregulation. Staggering progress was realized toward a better understanding of the genetic underpinnings and pathophysiology of many forms of atypical hemolytic uremic syndrome (aHUS) and C3-dominant glomerulopathies that are driven by complement system abnormalities. Many of these seminal discoveries paved the way for the design and characterization of several innovative therapies, some of which have already radically improved patients' outcomes. This review offers a broad overview of the exciting developments that have occurred in the recent past, with a particular focus on single-gene (or Mendelian), complement-driven aHUS and C3-dominant glomerulopathies that should be of interest to both nephrologists and kidney researchers. The discussion is restricted to genes with robust associations with both aHUS and C3-dominant glomerulopathies (complement factor H, complement component 3, complement factor H-related proteins) or only aHUS (complement factor B, complement factor I, and membrane cofactor protein). Key questions and challenges are highlighted, along with potential avenues for future directions.

Alternative pathway of complement activation has a beneficial role against Chandipura virus infection.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

Complement Component C3 and Complement Factor B Promote Growth of Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal keratinocytes (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Murine complement deficiency ameliorates acute cigarette smoke-induced nasal damage.

OBJECTIVE: Exposure to cigarette smoke is a risk factor for chronic rhinosinusitis. Current literature confirms complement fragments are activated in human nasal mucosa. The mechanism(s) responsible for this activation is unclear. We investigated the effects of cigarette smoke on nasal mucosa in vitro and via a model of cigarette smoke exposure by using animals deficient in complement components. STUDY DESIGN: Prospective, controlled animal and in vitro human cell line study. SETTING: University laboratory. SUBJECTS AND METHODS: Human respiratory epithelial cells were exposed to five, 10, and 20 percent cigarette smoke extract (CSE) in vitro in the presence or absence of human serum. Complement activation was assessed by enzyme-linked immunosorbent assay and immunofluorescent techniques. Complement-deficient (C3(-/-), n = 6; factor B(-/-), n = 50) and sufficient mice (wild type, n = 10) were exposed to the smoke of four cigarettes per exposure for two exposures per day for three days. Mice were sacrificed 12 hours after the last exposure, and the nasal cavity was surgically removed. Histological characteristics were analyzed by the use of a subjective scale and quantitative image analysis scoring systems. RESULTS: In vitro analysis of respiratory cell cultures demonstrated that exposure of serum to CSE resulted in complement activation. Furthermore, immunofluorescent staining for C3d could only be demonstrated in CSE-exposed cultures. In vivo analysis demonstrated that complement deficiency, either C3 or factor B deficiency, resulted in a significant reduction in histological evidence of damage as compared with wild-type control mice (wild type vs C3(-/-), P = 0.02; wild type vs factor B(-/-), P = 0.07; no significant difference between C3(-/-) vs factor B(-/-)). CONCLUSION: These data demonstrate that cigarette smoke activates the complement system. Furthermore, complement deficiency protected against smoke-induced mucosal damage in this small series.

Fragment Bb in amniotic fluid: evidence for complement activation by the alternative pathway in women with intra-amniotic infection/inflammation.

OBJECTIVE: Fragment Bb is an activator of the alternative pathway of the complement system. Recently, increased first trimester maternal plasma concentrations of this fragment were reported in patients destined to have a spontaneous preterm delivery before 34 weeks of gestation. The aim of this study was to determine whether the amniotic fluid (AF) concentrations of fragment Bb change with gestational age, spontaneous labor (term and preterm) and in the presence of intra-amniotic infection/inflammation (IAI). STUDY DESIGN: This cross-sectional study included patients in the following groups: (1) mid-trimester (n = 64); (2) term in spontaneous labor (n = 70); (3) term not in labor (n = 43); (4) spontaneous preterm labor (PTL) who delivered at term (n = 76); (5) PTL without IAI who delivered preterm (n = 73); (6) PTL with IAI (n = 76); (7) preterm prelabor rupture of membranes (PROM) without IAI (n = 71); and (8) preterm PROM with IAI (n = 71). Fragment Bb concentration in AF was determined by an enzyme-linked immunoassay. Non-parametric statistics were used for analyses. RESULTS: (1) Fragment Bb was detected in all AF samples (n = 544); (2) The median AF concentration of fragment Bb in patients at term not in labor was significantly higher than that of those in the mid-trimester [2.42 microg/ml, interquartile range (IQR) 1.78-3.22 vs. 1.64 microg/ml, IQR 1.06-3.49; p < 0.001]; (3) Among patients with PTL, those with IAI had a higher median AF fragment Bb concentration than that of woman without IAI, who delivered preterm (4.82 microg/ml, IQR 3.32-6.08 vs. 3.67 microg/ml, IQR 2.35-4.57; p < 0.001) and than that of women with an episode of PTL, who delivered at term (3.21 microg/ml, IQR 2.39-4.16; p < 0.001); (4) Similarly, among patients with preterm PROM, the median AF fragment Bb concentration was higher in individuals with IAI than in those without IAI (4.24 microg/ml, IQR 2.58-5.79 vs. 2.79 microg/ml, IQR 2.09-3.89; p < 0.001). (5) Among patients at term, the median AF fragment Bb concentration did not differ between women with spontaneous labor and those without labor (term in labor: 2.47 microg/ml, IQR 1.86-3.22; p = 0.97). CONCLUSIONS: (1) Fragment Bb, an activator of the alternative complement pathway, is a physiologic constituent of the AF, and its concentration increases with advancing gestational age; (2) AF concentrations of fragment Bb are higher in pregnancies complicated with IAI; and (3) labor at term is not associated with changes in the AF concentrations of fragment Bb. These findings suggest a role for fragment Bb in the host immune response against IAI.

[Association of polymorphism for porcine BF gene with reproductive traits and placental efficiency in Large White].

Taking BF gene as one of the candidate genes influencing on reproductive traits in present study, this study used PCR-RFLP analyzised the polymorphism of intron 1 of BF gene in Large White sows. A single nucleotide polymorphism (SNP) named as BF-intron 1-C79T was detected. And the three genotypes of CC, CT, TT got fitted Hardy-Weinberg equilibrium with chi2-fitness test. An association analysis was tested between the genetic polymorphism at intron 1 and total number born (TNB), number born alive (NBA), born weight (BW) and placental efficiency (PE) respectively. It showed that, at first parity, the TNB, NBA, BW and PE with CC genotype were higher than those with CT genotype but not significant (P>0.05). Meanwhile, in multiparous sow population, the TNB, NBA and PE with CC genotype were significantly more than those with TT genotype (P<0.05), with increase of 3.45, 3.92, and 23.80%, respectively. Thus BF-intron 1-C79T is suggested to be a potential genetic marker for the breeding on the reproductive traits and placental efficiency.

The alternative pathway of complement is activated in the glomeruli and tubulointerstitium of mice with adriamycin nephropathy.

The complement system effectively identifies and clears invasive pathogens as well as injured host cells. Uncontrolled complement activation can also contribute to tissue injury, however, and inhibition of this system may ameliorate many types of inflammatory injury. Several studies have demonstrated that the filtration of complement proteins into the renal tubules, as occurs during proteinuric renal disease, causes tubular inflammation and injury. In the present study, we tested the hypothesis that activation of the complement system in the urinary space requires an intact alternative pathway. Using a model of adriamycin-induced renal injury, which induces injury resembling focal segmental glomerulosclerosis, we examined whether mice deficient in factor B would be protected from the development of progressive tubulointerstitial injury. Complement activation was attenuated in the glomeruli and tubulointerstitium of mice with congenital deficiency of factor B (fB-/-) compared with wild-type controls, demonstrating that complement activation does occur through the alternative pathway. Deficiency in factor B did not significantly protect the mice from tubulointerstitial injury. However, treatment of wild-type mice with an inhibitory monoclonal antibody to factor B did delay the development of renal failure. These results demonstrate that complement activation in this nonimmune complex-mediated model of progressive renal disease requires an intact alternative pathway.

Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation.

Complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization: role of factor B and factor H.

The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.

Loss of complement activation and leukocyte adherence as Nippostrongylus brasiliensis develops within the murine host.

Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematode Nippostrongylus brasiliensis change as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage of N. brasiliensis is vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection, N. brasiliensis acquires the ability to largely avoid complement-dependent immune responses.

Cell surface activation of the alternative complement pathway by the fusion protein of measles virus.

Measles virus (MV)-infected cells are activators of the alternative human complement pathway, resulting in high deposition of C3b on the cell surface. Activation was observed independent of whether CD46 was used as a cellular receptor and did not correlate with CD46 down-regulation. The virus itself was an activator of the alternative pathway and was covered by C3b/C3bi, resulting in some loss in infectivity without loss of virus binding to target cells. The cell surface expression of MV fusion (F), but not haemagglutinin, envelope protein resulted in complement activation of the Factor B-dependent alternative pathway in a dose-dependent manner and F-C3b complexes were formed. The underlying activation mechanism was not related to any decrease in cell surface expression of the complement regulators CD46 and CD55. The C3b/C3bi coating of MV-infected cells and virus should ensure enhanced targeting of MV antigens to the immune system, through binding to complement receptors.

Lack of complement factor C3, but not factor B, increases hyperlipidemia and atherosclerosis in apolipoprotein E-/- low-density lipoprotein receptor-/- mice.

OBJECTIVE: To investigate the effect of complement deficiency on atherogenesis and lipidemia, we used mice deficient in the third complement component (C3-/-) or factor B (FB-/-). METHODS AND RESULTS: Complement-deficient mice were crossed with mice deficient in both apolipoprotein E and the low-density lipoprotein receptor (Apoe-/- LDLR-/-). The percent lesion area in the aorta at 16 weeks, determined by en face analysis, was 84% higher in C3-/- mice than in controls (11.8%+/-0.4% versus 6.4%+/-0.8%, mean+/-SEM, P<0.00005). The C3-/- mice also had 58% higher serum triglyceride levels (P<0.05) and a more proatherogenic lipoprotein profile, with significantly more low-density lipoprotein cholesterol and very-low-density lipoprotein triglycerides than control mice. The C3-/- mice weighed 13% less (P<0.01) and had a lower body fat content (3.5%+/-1.0% versus 13.1%+/-3.0%, P<0.01). There were no differences between FB-/- mice and controls. CONCLUSIONS: Complement activation by the classical or lectin pathway exerts atheroprotective effects, possibly through the regulation of lipid metabolism.

Critical role of the complement system in group B streptococcus-induced tumor necrosis factor alpha release.

Group B Streptococcus (GBS) is a major cause of newborn sepsis and meningitis and induces systemic release of tumor necrosis factor alpha (TNF-alpha), believed to play a role in morbidity and mortality. While previous studies have shown that GBS can induce TNF-alpha release from monocytes and macrophages, little is known about the potential modulating effect of plasma or serum on GBS-induced TNF-alpha release, and there are conflicting reports as to the host receptors involved. In a human whole-blood assay system, GBS type III COH-1 potently induced substantial monocyte TNF-alpha release in adult peripheral blood and, due to a higher concentration of monocytes, 10-fold-greater TNF-alpha release in newborn cord blood. Remarkably, GBS-induced TNF-alpha release from human monocytes was enhanced approximately 1000-fold by heat-labile serum components. Experiments employing C2-, C3-, or C7-depleted serum demonstrated that C3 activation via the alternative pathway is crucial for potent GBS-induced TNF-alpha release. Accordingly, whole blood from C3-deficient mice demonstrated significantly reduced GBS-induced TNF-alpha release. Preincubation with human serum enhanced the TNF-alpha-inducing activity of GBS in a C3- and factor B-dependent manner, implying deposition of complement components via the alternative pathway. GBS-induced TNF-alpha release was inhibited by monoclonal antibodies directed against each of the components of CR3 and CR4: the common integrin beta subunit CD18 and the alpha subunits CD11b (of CR3) and CD11c (of CR4). Blood derived from CR3 (CD11b/CD18)-deficient mice demonstrated a markedly diminished TNF-alpha response to GBS. We conclude that the ability of plasma and serum to greatly amplify GBS-induced TNF-alpha release reflects the activity of the alternative complement pathway that deposits fragments on GBS and thereby enhances CR3- and CR4-mediated monocyte activation.

Lack of a functional alternative complement pathway ameliorates ischemic acute renal failure in mice.

Ischemia/reperfusion (I/R) injury of the kidney is a common cause of acute renal failure (ARF) and is associated with high morbidity and mortality in the intensive care unit. The mechanisms underlying I/R injury are complex. Studies have shown that complement activation contributes to the pathogenesis of I/R injury in the kidney, but the exact mechanisms of complement activation have not been defined. We hypothesized that complement activation in this setting occurs via the alternative pathway and that mice deficient in complement factor B, an essential component of the alternative pathway, would be protected from ischemic ARF. Wild-type mice suffered from a decline in renal function and had significant tubular injury, particularly in the outer medulla, after I/R. We found that factor B-deficient mice (fB(-/-)) developed substantially less functional and morphologic renal injury after I/R. Furthermore, control wild-type mice had an increase in tubulointerstitial complement C3 deposition and neutrophil infiltration in the outer medulla after I/R, whereas fB(-/-) mice demonstrated virtually no C3 deposition or neutrophil infiltration. Our results demonstrate that complement activation in the kidney after I/R occurs exclusively via the alternative pathway, and that selective inhibition of this pathway provides protection to the kidneys from ischemic ARF.

Critical review of acylation-stimulating protein physiology in humans and rodents.

In the last few years, there has been increasing interest in the physiological role of acylation-stimulating protein (ASP). Recent studies in rats and mice, in particular in C3 (-/-) mice that are ASP deficient, have advanced our understanding of the role of ASP. Of note, the background strain of the mice influences the phenotype of delayed postprandial triglyceride clearance in ASP-deficient mice. Administration of ASP in all types of lean and obese mice studied to date, however, enhances postprandial triglyceride clearance. On the other hand, regardless of the background strain, ASP-deficient mice demonstrate reduced body weight, reduced leptin and reduced adipose tissue mass, suggesting that ASP deficiency results in protection against development of obesity. In humans, a number of studies have examined the relationship between ASP, obesity, diabetes and dyslipidemia as well as the influence of diet, exercise and pharmacological therapy. While many of these studies have small subject numbers, interesting observations may help us to better understand the parameters that may influence ASP production and ASP action. The aim of the present review is to provide a comprehensive overview of the recent literature on ASP, with particular emphasis on those studies carried out in rodents and humans.

Structure and flexibility of the multiple domain proteins that regulate complement activation.

In this review we summarise more than 10 years of biophysical exploration into the structural biology of the regulators of complement activation (RCA). The five human proteins responsible for regulation of the early events of complement are homologous and are composed largely from building blocks called "complement control protein (CCP) modules". Unlike most multiple domain proteins they do not contain any of the other widely occurring module types. This apparent simplicity of RCA structure, however, is belied by their sophistication of function. In fact, the structures of the individual CCP modules exhibit wide variations on a common theme while the extent and nature of intermodular connections is diverse. Some neighbouring modules within a protein stabilise each other and some co-operate to form specific binding surfaces. The degree of true "modularity" of CCPs is open to debate. The study of RCA proteins clearly illustrates the value of combining complementary structural biology techniques. The results could have implications for folding, evolution, flexibility and structure-function relationships of other molecules in the large, diverse and little understood category of multiple domain proteins.

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Major histocompatibility complex phenotypes influence serum testosterone concentration.

OBJECTIVES: (a) To confirm our earlier observation that the phenotype HLA-DR4,7 occurs with higher frequency in male patients with rheumatoid arthritis (RA) than in female patients. (b) To test the hypothesis that DR7 is associated with low normal serum testosterone (Te) levels in healthy males; this might explain the increased frequency of DR4,7 in male patients since there appears to be a relationship between low serum Te and RA. (c) To characterize the association between HLA alleles and serum Te concentration in healthy males. METHODS: An additional 82 Newfoundland (NF) RA patients were HLA-DR typed and, combined with our earlier data and data from the 11th International Histocompatibility Workshop, gave HLA-DR and sex information on 373 RA patients. Ninety-four healthy NF males were typed for HLA, the microsatellite marker TNFa (located close to the tumour necrosis factor alpha gene) and complement factor B (BF). An additional 38 males were included, selected partly based on their HLA-B type. RESULTS: We confirmed our earlier finding of a higher frequency of HLA-DR4,7 in male RA patients compared with female RA patients (P<0.01). Contrary to our expectations we found that DR7 was associated with higher than mean values of Te as were B5, B27, DR1, TNFa7 and BF F positivity. Conversely, low Te concentrations were found in men with B15, DR2, DR5, TNFa5 and who were BF F negative. In 28 male 'early-onset' RA patients we did not find an increased frequency of HLA alleles associated with low Te levels as compared with the frequency in 41 'late-onset' patients, suggesting that if low Te level is a risk factor and is present before onset of RA then the level cannot be explained by an association between Te level and major histocompatibility complex (MHC) phenotype. CONCLUSION: This study indicates that a man's MHC phenotype may influence his serum Te concentration, but the relationship of this, if any, to the pathogenesis of RA remains an area of speculation.