Circulating C3 Levels Research Articles

Liver factor B silencing to cure C3 glomerulopathy: Evidence from a mouse model of complement dysregulation

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease.Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely.Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury.

The complement component C3 is a fundamental plasma protein for host defense, produced largely by the liver. However, recent work has demonstrated the critical importance of tissue-specific C3 expression in cell survival. Here, we analyzed the effects of local versus peripheral sources of C3 expression in a model of acute bacterial pneumonia induced by Pseudomonas aeruginosa. Whereas mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient only in liver-derived C3 remained protected, comparable to wild-type mice. Human lung transcriptome analysis showed that secretory epithelial cells, such as club cells, express high levels of C3 mRNA. Mice with tamoxifen-induced C3 gene ablation from club cells in the lung had worse pulmonary injury compared with similarly treated controls, despite maintaining normal circulating C3 levels. Last, in both the mouse pneumonia model and cultured primary human airway epithelial cells, we showed that stress-induced death associated with C3 deficiency parallels that seen in Factor B deficiency rather than C3a receptor deficiency. Moreover, C3-mediated reduction in epithelial cell death requires alternative pathway component Factor B.Thus, our findings suggest that a pathway reliant on locally derived C3 and Factor B protects the lung mucosal barrier.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMβ2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Circulating C3 levels predict renal and global outcome in patients with renal vasculitis.

Several studies have demonstrated the crucial role of complement activation in the pathogenesis of ANCA-associated vasculitis. We aimed to assess the association between baseline serum C3 (sC3) levels and long-term outcomes in patients with renal vasculitis. This retrospective study included 111 patients with renal vasculitis from three hospitals who underwent a renal biopsy between 1997 and 2014. Serum levels of C3 were measured at the onset and the study population was divided into three tertiles according to sC3 concentrations (tertile 1 <106mg/dl; tertile 2 106-128mg/dl; tertile 3 >128mg/dl). Patients with lower sC3 (tertile 1) were compared with those having higher levels of sC3 (tertile 2 and tertile 3). Histological, clinical, and laboratory data were recorded for analysis. The primary end point was the composite of end-stage renal disease (ESRD) and death from any cause. Lower sC3 levels were associated with a higher need for dialysis and lower response rate to treatment (p=0.04 and p=0.007, respectively). Renal and global survival at 1 and 5years was 53 and 46% in patients with lower sC3 (tertile 1) compared with 72 and 65% in patients with higher sC3 (upper two tertiles) (p=0.04). In a multivariate Cox-regression model, when adjusted by renal function and histopatholologic categories, lower sC3 remained as an independent predictor of ESRD and death (HR, 1.9; 95% CI, 1.1 to 3.4; p=0.02). Baseline serum C3 levels have an independent prognostic value in predicting long-term renal and global survival in patients with renal vasculitis.

Atypical Hemolytic Uremic Syndrome and Chronic Ulcerative Colitis Treated with Eculizumab.

BackgroundHemolytic-uremic syndrome (HUS) presents with hemolytic anemia, thrombocytopenia, and thrombotic microangiopathy of the kidney and usually results from Shiga-toxin induced activation of the alternative complement pathway. Gastroenteritis is a common feature of the Shiga-toxin producing Escherichia coli HUS, referred to as STEC-HUS. An inherited or acquired complement dysregulation may lead to HUS referred to as non-STEC or atypical (a)HUS. Although gastroenteritis is not a common presentation of aHUS, some patients develop ischemic colitis and may be misdiagnosed as acute appendicitis or acute ulcerative colitis (UC).Case Diagnosis –TreatmentWe present a patient with low circulating complement (C) 3 levels who developed aHUS in the course of chronic active UC. Resolution of renal and gastrointestinal manifestations in response to treatment with eculizumab, a humanized monoclonal antibody against terminal C5 protein suggests the role of alternative complement in the pathogenesis of both, aHUS and UC.ConclusionThis case illustrates that dysregulation of the alternative complement pathway may manifest in other organs besides the kidney and that the circulating C3 levels do not correlate with the disease activity or the clinical response to eculizumab.

Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy.

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN.

Complement protein C3 and coronary artery calcium in middle-aged women with polycystic ovary syndrome and controls

Circulating complement protein C3 (C3) levels have been associated with coronary artery calcification (CAC) in women with systemic lupus erythematosus, but have yet to be evaluated in women with polycystic ovary syndrome (PCOS). We aimed to determine whether C3 levels were elevated in women with PCOS compared to controls and to quantify the association of C3 with cardiovascular disease (CVD) risk factors and CAC and if PCOS modified this association. This cross-sectional analysis included 132 women with PCOS and 155 controls, 35–62 years old, from the third visit of a case-control study. CAC was measured during the study visit, and circulating C3 was measured in stored sera. The presence of CAC and CAC categories (Agatston score 0, 1–9.9 and ≥10) were used for logistic and ordinal regression analysis, respectively. C3 levels were not significantly different between women with PCOS and controls. Among all women, C3 was associated with the presence of CAC and increasing CAC groups after adjusting for age, PCOS status and insulin or body mass index (BMI), all p < 0.05. In addition, C3 was associated with the presence of CAC after adjusting for age, PCOS status, BMI, insulin and African American race, p = 0.049. PCOS status did not modify these associations. In conclusion, circulating C3 levels may prove beneficial in identifying women at risk of CVD in women with PCOS and the general population.

Distinct roles for complement in glomerulonephritis and atherosclerosis revealed in mice with a combination of lupus and hyperlipidemia

Objective Although the accelerating effect of systemic lupus erythematosus (SLE) on atherosclerosis is well established, the underlying mechanisms are unknown. The aim of this study was to explore the hypothesis that lupus autoimmunity modulates the effect of hypercholesterolemia in driving arterial pathologic development.>Methods Low-density lipoprotein receptor–deficient (Ldlr−/−) mice were crossed with B6.129-Sle16 (Sle16)–congenic autoimmune mice to obtain Sle16. Ldlr−/− mice, which were compared with Ldlr−/− and Sle16 control mice. All mice were fed either a low-fat or high-fat diet. Groups of mice were compared, by strain and by diet group, for features of accelerated atherosclerosis and autoimmunity.Results Presence of the Sle16 locus significantly increased the extent of atherosclerosis in Ldlr−/− mice. Circulating C3 levels were significantly reduced in Sle16.Ldlr−/− mice compared to Ldlr−/− control mice and this was paralleled by a marked reduction in arterial lesion C3 deposition despite similar levels of IgG deposition between the groups. Increased numbers of apoptotic cells in plaques were observed in the high-fat–fed Sle16.Ldlr−/− mice, consistent with the observed defective clearance of cellular debris. After receiving the high-fat diet, Sle16.Ldlr−/− mice developed glomerulonephritis and displayed enhanced glomerular C3 deposition.Conclusion These results indicate that accelerated atherosclerosis and renal inflammation in SLE are closely linked via immune complex formation and systemic complement depletion. However, whereas hyperlipidemia will enhance renal immune complex–mediated complement activation and the development of nephritis, accelerated atherosclerosis is, instead, related to complement depletion and a reduction in the uptake of apoptotic/necrotic debris. These results suggest that aggressive treatment of hyperlipidemia in patients with SLE may reduce the occurrence of lupus nephritis, as well as diminish the risk of accelerated atherosclerosis.

Decreased Circulating C3 Levels and Mesangial C3 Deposition Predict Renal Outcome in Patients with IgA Nephropathy

Background and AimsMesangial C3 deposition is frequently observed in patients with IgA nephropathy (IgAN). However, the role of complement in the pathogenesis or progression of IgAN is uncertain. In this observational cohort study, we aimed to identify the clinical implications of circulating C3 levels and mesangial C3 deposition and to investigate their utility as predictors of renal outcomes in patients with IgAN.MethodsA total of 343 patients with biopsy-proven IgAN were enrolled between January 2000 and December 2008. Decreased serum C3 level (hypoC3) was defined as C3 <90 mg/dl. The study endpoint was end-stage renal disease (ESRD) and a doubling of the baseline serum creatinine (D-SCr).ResultsOf the patients, there were 66 patients (19.2%) with hypoC3. During a mean follow-up of 53.7 months, ESRD occurred in 5 patients (7.6%) with hypoC3 compared with 9 patients (3.2%) with normal C3 levels (P = 0.11). However, 12 patients (18.2%) with hypoC3 reached D-SCr compared with 17 patients (6.1%) with normal C3 levels [Hazard ratio (HR), 3.59; 95% confidence interval (CI), 1.33–10.36; P = 0.018]. In a multivariable model in which serum C3 levels were treated as a continuous variable, hypoC3 significantly predicted renal outcome of D-SCr (per 1 mg/dl increase of C3; HR, 0.95; 95% CI, 0.92–0.99; P = 0.011). The risk of reaching renal outcome was significantly higher in patients with mesangial C3 deposition 2+ to 3+ than in patients without deposition (HR 9.37; 95% CI, 1.10–80.26; P = 0.04).ConclusionsThis study showed that hypoC3 and mesangial C3 deposition were independent risk factors for progression, suggesting that complement activation may play a pathogenic role in patients with IgAN.

Effect of weight loss on C3 and C4 components of complement in obese patients

Circulating C3 levels are elevated in obese patients, but how this factor is affected after weight loss through diet is a question that is yet unanswered. Therefore, the aim of this study was to evaluate the effects of weight loss on lipid and hydrocarbonated metabolism parameters and on the levels of C3 and C4 components of complement in obese patients. This is a longitudinal intervention study based on a 6-week very low-calorie diet (VLCD), a liquid formula of 603 kcal/day. A total of 131 middle-aged patients were distributed among grades II, III and IV of obesity. Anthropometric parameters, total cholesterol, triglycerides, high-density lipoprotein cholesterol, LDLc, apolipoproteins A-I and B-100, glucose, insulin, HOMA-IR and C3 and C4 levels were evaluated at baseline and after 6 weeks of intervention. After VLCD, the moderate weight loss was accompanied by a significant reduction in C3 levels in grade III and grade IV patients (10.2% and 15.4%, respectively; P < 0.001). C4 levels were not altered. Adherence to the diet improved anthropometric parameters and was accompanied by a significant decrease in all lipid profile parameters (P < 0.001). In addition, weight loss was associated with an improvement in hydrocarbonated metabolism as shown by the decrease in glucose levels and HOMA-IR (P < 0.01). Our findings show that in severely obese patients following a VLCD for 6 weeks produces reductions in factor C3, a biomarker of cardiovascular disease, and a significant improvement in some features of metabolic syndrome. In this way, the abovementioned diet may represent an effective strategy for treating obesity and related cardiovascular risk factors.

Membranoproliferative Glomerulonephritis with Isolated C3 Deposits: Case Report and Literature Review

Membranoproliferative glomerulonephritis with isolated C3 deposits (MPGNC3) is an uncommon condition characterized by overt glomerular C3 deposits in the absence of immunoglobulins and intramembranous dense deposits. Here the authors describe the clinical and morphological features of primary MPGNC3 in a 13-year-old boy and critically review the previously published cases. The patient presented with nephrotic syndrome and microscopic hematuria. Blood tests revealed very low circulating C3 levels. The renal biopsy exhibited subendothelial, subepithelial, and mesangial deposits, with C3 but not immunoglobulins seen on immunofluorescence. This case and the review of the literature indicate that the serum complement profile with decreased levels of C3 and normal levels of classical pathway components together with glomerular deposits containing exclusively complement C3 is highly suggestive of alternative pathway activation. The diagnosis of acquired and/or genetic complement abnormalities in some cases supports that complement dysregulation is implicated in the pathogenesis of MPGNC3. Such data show great promise to provide new therapy strategies based on modulation of the complement system activity.

Mesangial cell complement receptor 1‐related protein y limits complement‐dependent neutrophil accumulation in immune complex glomerulonephritis

The absence of complement receptor 1 (CR1) related gene/protein y (Crry) leads to embryonic lethality as a result of unrestricted complement activation and concomitant neutrophil infiltration. Here we used Crry(-/-)C3(+/-) mice to investigate the role of Crry in the pathogenesis of immune complex glomerulonephritis (GN). After 3 weeks of immunization with horse spleen apoferritin, six of nine Crry(-/-) C3(+/-) mice and none of the six control C3(+/-) mice developed proliferative GN (P = 0.010). After 5 weeks of immunization, GN scores in Crry(-/-) C3(+/-) mice were 0.67 +/- 0.22 mean +/- standard error of the mean (SEM), compared with 0.32 +/- 0.16 in C3(+/-) mice. Glomerular hypercellularity was attributable to neutrophil infiltration in mice with GN (1.7 +/- 0.3/glomerulus) compared with those without GN (0.4 +/- 0.1/glomerulus) (P = 0.001). Absent staining for alpha-smooth muscle actin and proliferating cell nuclear antigen suggested that mesangial cell proliferation did not play a significant role in this model. Serum C3 levels in Crry(-/-) C3(+/-) mice were approximately 20% and 30% those of wild-type mice and C3(+/-) mice, respectively. To determine whether this acquired hypocomplementaemia was relevant to this GN model system, Crry(-/-) C3(+/-) mouse kidneys were transplanted into wild-type mice followed by immunization with apoferritin for 1 or 2 weeks. Surprisingly, none of the Crry(-/-) C3(+/-) mouse kidneys developed GN at these early time-points, indicating that increasing circulating C3 levels several-fold did not increase susceptibility to GN. Renal expression of decay-accelerating factor was not different among any of the groups studied. Thus, our data indicate that mesangial cell Crry limits complement activation and subsequent neutrophil recruitment in the setting of local immune complex deposition.

Extrahepatic secreted complement C3 contributes to circulating C3 levels in humans.

The majority of complement protein C3 is synthesized by the liver, but many other cell types produce small amounts of functionally active C3. The overall contribution of such extrahepatic C3 production to the total circulating C3 level is unknown. Bone marrow and extrahepatic C3 productions were quantified in bone marrow transplant (BMT) and liver transplant (LT) recipients, respectively, where a mismatch for the C3 allotypes distinguished by the mAb HAV 4-1 had occurred. In the BMT group, donor-derived C3 was detected by ELISA and immunoblotting techniques. It contributed to 0.1 to 2.6% of the total circulating C3 during the immediate post-BMT period in response to inflammatory stimuli. Cell culture and immunostaining techniques demonstrated that monocytes were the source of the C3. By 6 wk following BMT, donor-derived C3 levels had decreased to below the detection limit of the assays. By contrast in the LT group, total extrahepatic C3 levels were higher (3.1-5.7% of the total circulating C3) and remained stable for up to 1 yr post-LT. This study demonstrates that extrahepatic derived C3 forms a larger proportion of the circulating C3 levels than was considered previously and that in the resting state, most of this extrahepatically derived C3 comes from nonmyeloid sources. In addition, monocytes, which in the resting state contribute negligible amounts of C3, have the potential when stimulated to contribute significantly to the total systemic C3 pool. These findings highlight the importance of locally secreted complement proteins.

C6 depletion reduces proteinuria in experimental nephropathy induced by a nonglomerular antigen.

The role of the complement membrane attack complex, C5b-9, in mediating glomerular injury has been well defined in models of membranous nephropathy induced by antibody to endogenous glomerular epithelial cell membrane antigens. The effect of selective C6 depletion (to prevent C5b-9 formation) on morphologic characteristics and proteinuria in a model of in situ subepithelial immune complex nephritis induced by an exogenous cationized antigen (human immunoglobulin G (IgG)) followed by rabbit antibody to human IgG was studied. Selective C6 depletion was achieved by repeated administration of a goat antibody to rat C6. Other groups were treated with cobra venom factor to induce generalized complement depletion and with sublethal irradiation to deplete circulating leukocytes. In C6-depleted rats, C6 levels were reduced to less than 3% of baseline throughout the 2 days of the study compared with over 100% in controls. At 4 h after disease induction, glomerular deposition of antigen and antibody were similar in C6D and control groups by immunofluorescence and by direct measurement of glomerular deposition of radiolabeled antigen and antibody (cationized 131I human IgG, 9.1 +/- 0.1 micrograms/38,000 glomeruli in C6D versus 9.8 +/- 0.9 in controls; P = was not significant; rabbit 125I-labeled anti-human IgG, 104 +/- 10 ng in C6D versus 80 +/- 9 ng in controls; P = was not significant). Circulating C3 levels and glomerular C3 deposition were also similar in C6D and control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement (C3) levels and the effect of C3 depletion in infections of Trypanosoma brucei in mice

Summary Circulating C3 levels and parasitaemias have been measured in four groups of mice chronically infected with Trypanosoma brucei: group A (normal); group B (irradiated, reconstituted); Group C (T cell‐deprived); and group D (T cell‐deprived, C3‐depleted by treatment with cobra venom factor). In groups A‐C, C3 levels first rose two to three times normal, C3 thus behaving as an acute phase reactant. Three weeks after infection C3 returned to normal levels for the remainder of the infection. It is thus unlikely that the severe generalized immunodepression seen in mice infected with this trypanosome, is in any way dependent upon a reduction of circulating C3. The curves of parasitaemia in all four groups of mice were essentially similar, even though in group D mice, C3 levels were reduced to about 10% of normal for the first 3 weeks of infection. From this finding, it is argued that complement has no essential role in the mechanisms whereby mice control successive variant populations of T. brucei in the blood. Variant‐specific IgM antibodies, acting as trypagglutinins, are probably all that are required to control blood stream infections.

Role of complement in endotoxin initiated lethality in mice

We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice. The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality. The complement system in both strains was demonstrated to be responsive in vitro to activation both by E. coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S. minnesota R595 LPS, which activates almost exclusively the classical pathway. In vivo injection of high (lethal) doses of E. coli 0111:B4 LPS I and S. minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr. In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E. coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation. In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice.