An analysis of functional activity via the three complement pathways during hemodialysis sessions: a new insight into the association between the lectin pathway and C5 activation.

BACKGROUND: We have recently demonstrated that hemodialysis (HD) patients have significantly higher levels of functional complement activity (FCA) in all three pathways, i.e. the classical pathway, alternative pathway and lectin pathway (LP), than in age-matched controls, though the role of FCA during HD still remains unknown. METHODS: Serial plasma or serum samples were obtained from five patients during HD in order to investigate the kinetics of complement components. The levels of the C5b-9 complex, the FCA of the three pathways, a derivative of C3a (C3a desArg) and a derivative of C5a (C5a desArg) in the samples were analyzed. RESULTS: The levels of the C5b-9 complex at 60 min were significantly increased when compared with those at 0 min. Functional activities for all three pathways showed different patterns so the same tendency between pathways was not observed. The levels of C3a desArg and C5a desArg at 60 min were markedly increased when compared with those at 0 min. A Spearman's rho test showed a strong positive correlation between functional LP activity and C5a desArg. CONCLUSIONS: These findings lead to new insights into the FCA during HD and suggest that functional LP activity has an important role in C5 activation.

Excretion of complement proteins and its activation marker C5b-9 in IgA nephropathy in relation to renal function.

BACKGROUND: Glomerular damage in IgA nephropathy (IgAN) is mediated by complement activation via the alternative and lectin pathways. Therefore, we focused on molecules stabilizing and regulating the alternative pathway C3 convertase in urine which might be associated with IgAN pathogenesis. METHODS: Membrane attack complex (MAC), properdin (P), factor H (fH) and Complement receptor type 1 (CR1) were quantified in urine samples from 71 patients with IgAN and 72 healthy controls. Glomerular deposition of C5, fH and P was assessed using an immunofluorescence technique and correlated with histological severity of IgAN and clinical parameters. Fibrotic changes and glomerular sclerosis were evaluated in renal biopsy specimens. RESULTS: Immunofluorescence studies revealed glomerular depositions of C5, fH and P in patients with IgAN. Urinary MAC, fH and P levels in IgAN patients were significantly higher than those in healthy controls (p < 0.001), but CR1 was significantly lower than that in healthy controls (p < 0.001). Urinary MAC and fH levels were positively correlated with serum creatinine (sCr), urinary N-acetyl-ß-D-glucosaminidase (u-NAG), urinary ß2 microglobulin (u-Bm), urinary protein (p < 0.001), interstitial fibrosis (MAC: p < 0.01, fH: p < 0.05) and the percentage of global glomerular sclerosis (p < 0.01). Urinary P was positively correlated with u-NAG, u-Bm, and urinary protein (p < 0.01). CONCLUSIONS: Complement activation occurs in the urinary space in IgAN and the measurement of levels of MAC and fH in the urine could be a useful indicator of renal injury in patients with IgAN.

Low CR1 (C3b receptor) level on erythrocytes is associated with poor prognosis in hemodialysis patients.

BACKGROUND: Erythropoietin in patients under dialysis treatment for renal failure is low which induces anemia. Treatment with recombinant erythropoietin (rEPO) has been used routinely as a supplement treatment for these patients. Immune complexes (IC) react with complement and bind to CR1 on erythrocytes (E-CR1), and are transported to the liver and/or spleen where IC removal and degradation occurs. The erythrocytes then return to circulation where they bind to additional IC. There are some patients whose E-CR1 expression is low with chronic anemia in spite of rEPO treatment. We hypothesized that in hemodialysis (HD) patients altered host defense against infection is associated with low levels of E-CR1. We examined if low E-CR1 in dialysis patients constitutes a risk factor for reduced host defense and poor outcome. METHODS: In 95 HD patients, E-CR1 was quantified using a monoclonal E-CR1 antibody and FACS analysis followed by clinical course studies for 5 years. RESULTS: The patients were divided into three groups by E-CR1 level. Percent survival for the low E-CR1 group (53.3%) was significantly lower than the high E-CR1 group (86.4%) (p < 0.01). There were more hepatitis C virus-positive patients within the low E-CR1 group (27.3%) than in the high E-CR1 group (4.7%) (p < 0.05). Furthermore, 10 patients with the lowest E-CR1 levels had severe complications, notably infection at an arteriovenous fistula. CONCLUSION: A reduced E-CR1 level might be a risk factor for reduced host defense and can be used as a predicting factor for poor prognosis in a HD patient.

Hypercomplementemia in adult patients with IgA nephropathy.

IgA nephropathy (IgAN) is the most common form of chronic glomerulonephritis. Although glomerular deposition of complement components is well known, the evidence of serological complement activation in IgAN is inconclusive. We hypothesized that serum levels of complement components and regulatory proteins in patients with IgAN are correlated with its pathogenesis. In the present study we measured complement components in 50 patients with IgAN and 50 healthy volunteers. C5, C1 inhibitor, factor B, C4 binding protein, factor H, and factor I were measured with the use of single radial immunodiffusion. Mannose-binding lectin (MBL) and properdin (P) were measured by enzyme-linked immunosorbent assay (ELISA). The correlations among complements in the sera of patients with clinical gradings for IgAN (i.e., the good prognosis group, relatively good prognosis group, relatively poor prognosis group, and poor prognosis group) were evaluated. CH50, C4, factor B, P, factor I, and factor H were significantly higher in IgAN patients than in healthy controls. There were significant correlations between C5 and C4 binding protein, between C3 and C5, or between C4 and factor B in patients with IgAN. In the poor prognosis group, C4 binding protein was significantly higher than in the other groups of IgAN patients. hypercomplementemia occurs in IgAN and is associated with an increase in complement regulatory protein (CRP). C4 binding protein analyses can be used to predict disease prognosis.

Acquired loss of erythrocyte complement receptor type 1 in patients with diabetic nephropathy undergoing hemodialysis.

BACKGROUND: Complement receptor type 1 on erythrocytes (E-CR1) plays important roles not only in the regulation of complement activation, but also the clearance of immune complexes. Reduced E-CR1 was previously found in patients undergoing hemodialysis (HD). We investigated whether the E-CR1level in HD patients with diabetic nephropathy (DMN) is decreased. The levels of decay accelerating factor (DAF) and CD59 on erythrocytes (E) were also determined to ascertain whether the loss of CR1 is a specific phenomenon or other complement regulatory proteins are also affected. METHODS: The levels of CR1, DAF, and CD59 on E were analyzed in 176 HD patients with DMN, 101 HD patients with non-diabetes mellitus renal diseases (non-DMN), and 108 healthy individuals. Hind III restriction fragment length polymorphism of intron 27 of the CR1 gene was analyzed. The serum-soluble CR1 levelwas measured by ELISA. RESULTS: The E-CR1 level was significantly lower in the DMN group than the non-DMN group (p < 0.0001) and healthy individuals (p < 0.05). The E-CR1 level was significantly higher in the non-DMN group than in healthy individuals (p < 0.01). The levels of E-DAF and E-CD59 were significantly lower in the DMN group than non-DMN group (DAF, p < 0.01; CD59, p < 0.0001). Within each genotype of the CR1 gene, the E-CR1 level was significantly lower in the DMN group than in the non-DMN group and healthy individuals (non-DMN, p < 0.01; healthy individuals, p < 0.05). The serum-soluble CR1 level was significantly higher in the DMN group than non-DMN group and control group (p < 0.01 each). However, soluble CR1 did not correlate with E-CR1. CONCLUSION: Acquired loss of E-CR1 was found among HD patients with DMN. From the viewpoint of host defense, it may be a prognostic factor.

Glomerular deposition and urinary excretion of complement factor H in idiopathic membranous nephropathy.

BACKGROUND/AIMS: The complement system plays an important role in the pathogenesis of membranous nephropathy (MN). In order to elucidate the regulatory mechanism of complement activation, we demonstrated glomerular deposition and urinary excretion of complement factor H, which controls the alternative pathway and the amplification loop at the C3 step, in patients with idiopathic MN. METHODS: Renal biopsy specimens from 20 patients with idiopathic MN were studied immunohistochemically using monoclonal antibodies against complement components including factor H. SDS-PAGE and Western blotting analysis of urine samples were performed, and the urinary excretion of factor H and C5b-9 were measured by quantitative sandwich ELISA. RESULTS: Intense glomerular deposition of factor H was observed with C3b.C3c and C5b-9 at an early stage of the disease. Factor H was detected in Western blots of urine samples, but factor H-like protein 1 (FHL-1) was not. The mean level of urinary factor H was elevated (86.30 +/- 21.93 U/mg urinary creatinine) in comparison to that of normal controls (4.76 +/- 1.03 U/mg urinary creatinine). Urinary factor H level exhibited no correlation with clinical parameters; however, a negative correlation was found between urinary C5b-9/factor H and creatinine clearance (r = 0.662, p < 0.01). CONCLUSION: The source of glomerular and urinary factor H is supposedly a 150-kD protein. There was no evidence to suggest that FHL-1 is synthesized at the site of inflammation. The urinary C5b-9 to urinary factor H ratio is indicative of the degree of ongoing complement activation in the glomeruli and complement-mediated renal injury. These findings suggest that factor H contributes to the control mechanism of in situ complement activation and prevents renal damage in idiopathic MN.

Quantitative polymorphism of complement receptor type 1 (CR1) in patients undergoing haemodialysis.

BACKGROUND: The level of complement receptor type 1 (CR1) on erythrocytes (E-CR1) is determined by the presence of high (H) or low (L) expression alleles. We investigated whether acquired loss of E-CR1 occurs in haemodialysis patients and, if so, which factors may contribute to acquired loss of E-CR1 in these patients. METHODS: The E-CR1 level was determined in 195 Japanese haemodialysis patients, and we selected patients with a high or low E-CR1 level. In patients with low E-CR1 expression, sequence analysis of polymorphic sites (A3650G and C5507G) in the CR1 gene was performed. To assess the effect of the type of dialysis membrane used in the patients with low E-CR1 expression, the dialysis membrane was changed from a cellulose membrane to a biocompatible membrane (to a polyacrylonitrile membrane and then to a polysulfone membrane). To evaluate the susceptibility of E-CR1 to proteolysis, erythrocytes were incubated with various concentrations of trypsin, and the level of remaining CR1 on the erythrocytes was determined. RESULTS: Among patients with high E-CR1 expression (n = 30), 87% had HH alleles and 13% had HL alleles. Among patients with low E-CR1 expression (n = 29), 24% had LL alleles, 45% had HL alleles and 31% had HH alleles. Nucleotides 3650G and 5507G in the CR1 gene were associated with the L allele. Nucleotides 3650A and 5507C were associated with the H allele. Only one patient with HH alleles had nucleotides 3650G and 5507C. Three months after changing the haemodialysis membrane, the E-CR1 level significantly increased (P<0.02). The proteolysis curves of E-CR1 of patients with low or high E-CR1 expression and normal controls were similar. CONCLUSION: Use of a non-biocompatible dialysis membrane may contribute to acquired loss of E-CR1 in haemodialysis patients.

Recombinant EPO therapy increases erythrocyte expression of complement regulatory proteins.

BACKGROUND: One of the complications of hemodialysis (HD) therapy is anemia caused by erythropoietin (EPO) deficiency. Recombinant EPO (rEPO) has been used routinely as a supplemental treatment. Erythrocyte expression of the complement regulatory proteins decay accelerating factor (DAF) and CD59 restricts complement activation and inhibits hemolysis. We hypothesized that the efficacy of rEPO treatment may be caused in part by the ability of rEPO to increase erythrocyte expression of DAF and CD59. METHODS: DAF, CD59, and complement receptor 1 (CR1) levels were analyzed for a group of 95 HD patients and compared with those of a control group. To evaluate effects of discontinuation of rEPO therapy, rEPO therapy was stopped for 12 HD patients until hematocrits decreased to less than 25%. DAF and CD59 levels then were reanalyzed. RESULTS: In the 95 HD patients, three factors correlated significantly: DAF and CD59 (r = 0.642), DAF and CR1 (r = 0.503), and CD59 and CR1 (r = 0.324), whereas no correlations were found in the group of 42 healthy controls. In the experiment in which rEPO therapy was discontinued, 8 of 12 patients reached the defined level of anemia 4 to 7 weeks after rEPO treatment had been withheld. Both DAF and CD59 levels decreased significantly after discontinuation of rEPO therapy (P < 0.01). DAF and CD59 levels increased in all 8 patients after rEPO treatment was reinitiated (P < 0.01), and CR1 levels increased in 5 of 8 patients. Four of 12 patients showed no evidence of anemia after discontinuation of rEPO treatment. In these patients, DAF, CD59, and CR1 levels did not change before or after withholding rEPO therapy. CONCLUSION: One of the mechanisms mediating the efficacy of EPO therapy is increased DAF and CD59 expression.

Urinary complement factor H in renal disease.

BACKGROUND: Complement factor H (hCFH) plays a key inhibitory role in the control of the alternative complement pathway. We examined whether urinary hCFH (U-hCFH) levels is useful as an indirect indicator of renal damage. METHODS: Urine samples were obtained from 104 patients with renal disease. Urine was collected with 10 mM EDTA and U-hCFH levels were measured using the BTA TRAK Assay Kit. RESULTS: In the 62 patients with nephritis, the levels of U-hCFH were elevated (range 15-52,198 U/ml) over the normal range (0-14 U/ml). U-hCFH levels of patients with chronic renal failure, lupus nephritis, membranoproliferative glomerulonephritis, focal glomerulosclerosis were higher than that of IgA nephropathy patients (p < 0.05). In the patients with minimal change disease, showed high levels of U-hCFH during the nephrotic syndrome. U-hCFH was correlated significantly with urinary protein and urinary N-acetyl-beta-D-glucosaminidase. CONCLUSIONS: We demonstrated that U-hCFH was detected in the urine of nephritis patients.