Kisara Onda

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

BackgroundGlomerular 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.MethodsMembrane 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.ResultsImmunofluorescence 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-β-D-glucosaminidase (u-NAG), urinary β2 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).ConclusionsComplement 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.

Fluctuation of serum C3 levels reflects disease activity and metabolic background in patients with IgA nephropathy.

BACKGROUND We focused on the fluctuations of serum C3 levels throughout the clinical course of patients and investigated the relationship between these fluctuations and clinical findings. METHODS IgA nephropathy patients (n = 122) were enrolled in the present study. Serum C3 and other clinical markers were compared at the time of renal biopsy and at last follow-up (6.67 ± 2.07 years). Patients were divided into 3 groups based on serum C3 levels: Group I with first C3 levels below the mean -1 SD, which turned into an increase at last observation; group II with first C3 levels more than the mean +1 SD, which turned into a decrease at last observation; and group III, with first C3 levels more than the mean +1 SD, which turned into an increase at last observation. First and last levels of clinical markers were compared among the 3 groups. RESULTS Serum C3 levels of the patients whose renal symptoms, including hematuria, proteinuria and estimated glomerular filtration rate (eGFR), were improved, were significantly increased at last observation (p<0.05, p<0.01, p<0.01, respectively). Age, total cholesterol and triglyceride levels in group III were significantly higher than those in group I. Group II showed a significant reduction of urinary protein. Groups I and II maintained renal function, but group III showed a significant deterioration of renal function. CONCLUSIONS The levels and fluctuations of serum C3 might reflect the disease activity and metabolic alteration in patients with IgA nephropathy.

Serum Concentration of Complement Components of the Lectin Pathway in Maintenance Hemodialysis Patients, and Relatively Higher Levels of L-Ficolin and MASP-2 in Mannose-Binding Lectin Deficiency

Mannose‐binding lectin (MBL), L‐ficolin and MBL associated serine protease‐2 (MASP‐2) are molecules involved in initiation of the lectin pathway (LP) in the complement system. Although MBL deficiency is observed in almost 10% of healthy people, studies of associations between MBL deficiency and end‐stage renal disease (ESRD) remain rare. The objective of the present study is to clarify the significance of the LP in maintenance hemodialysis (HD) patients, especially in terms of MBL levels. Two hundred and forty‐four HD patients who had been followed up for 74 ± 84 months and 199 healthy controls were included in this study. Measurements of serum concentrations of MBL, L‐ficolin, and MASP‐2 were performed. Low serum MBL levels (<0.1 µg/mL) in the patients were confirmed by examination of a point mutation in the Mbl‐2 gene. Seventeen HD patients (7%) and 20 healthy controls (10%) had MBL deficiency. During the follow‐up period, 99 patients died. There was no significant difference in the frequency of deaths by infectious diseases between MBL deficient and non‐deficient patients. In both patients and healthy controls with MBL deficiency, the serum concentration of L‐ficolin tended to be high, and that of MASP‐2 was significantly high (P < 0.05). MBL deficiency is not a risk factor for HD induction or life‐threatening infections. It is postulated that the elevation of concentration of the two components of the LP, L‐ficolin and MASP‐2, may compensate for the insufficient activity of the LP in MBL deficiency.

Risk of overestimation of kidney function using GFR-estimating equations in patients with low inulin clearance.

Accurate estimation of the glomerular filtration rate (GFR) is very important in clinical practice. Although renal inulin clearance (Cin) is the gold standard for measuring GFR, the procedure for Cin measurement is complicated. Use of GFR‐estimating equations has been increasing recently due to their simplicity. The objectives of the present study are to analyze the correlation between Cin and other GFR‐estimating parameters and to investigate their clinical usefulness and limitation.

Metabolic impact on serum levels of complement component 3 in Japanese patients.

The aim of this study was to explore the association between the serum concentration of complement component 3 (C3) and a variety of metabolic parameters. The study involved 125 patients in our outpatient clinic. Anthropometric and clinical laboratory data were collected and statistical associations between the serum concentration of C3 and other parameters were evaluated in a cross‐sectional as well as a prospective manner. A group of male patients with metabolic syndrome (Mets, n=35) were characterized by marked increase in serum concentrations of C3, body mass index (BMI), waist circumference, hemoglobin (Hb) A1c, insulin resistance (HOMA‐IR), triglyceride, uric acid, urinary protein, and Hb. In a one‐way analysis of variance of all subjects, the serum concentration of C3 was significantly elevated as the number of items of complying with the Mets diagnostic criteria increased. In 60 of 125 patients who did not have diabetes and were given anti‐lipogenetic medication, the serum concentration of C3 showed significant positive associations with serum levels of CH50, insulin, HOMA‐IR, total cholesterol, hematocrit, LDL‐c, C4, Hb, triglyceride, BMI, and albumin. In a prospective follow‐up evaluation (n=35), there was a significant positive association between ΔC3 (the second concentration of serum C3 minus the first concentration of serum C3)and ΔHOMA‐IR (the second concentration of HOMA‐IR minus the first concentration of HOMA‐IR). In conclusion, in Japanese patients, there is evidence implicating C3 concentration as a marker of Mets coinciding with insulin resistance. J. Clin. Lab. Anal. 24:113–118, 2010.

Complement in patients receiving maintenance hemodialysis: functional screening and quantitative analysis

BackgroundThe complement system is vital for innate immunity and is implicated in the pathogenesis of inflammatory diseases and the mechanism of host defense. Complement deficiencies occasionally cause life-threatening diseases. In hemodialysis (HD) patients, profiles on complement functional activity and deficiency are still obscure. The objectives of the present study were to measure the functional complement activities of the classical pathway (CP), lectin pathway (LP) and alternative pathway (AP) using a novel method and consequently to elucidate the rates of deficiencies among HD patients.MethodsIn the present study, 244 HD patients at one dialysis center and 204 healthy controls were enrolled. Functional complement activities were measured simultaneously using the Wielisa®-kit. The combination of the results of these three pathway activities allows us to speculate which candidate complement is deficient; subsequently, the deficient complement was determined.ResultsAll three functional complement activities were significantly higher in the HD patients than in the control group (P < 0.01 for all cases). After identifying candidates in both groups with complement deficiencies using the Wielisa®-kit, 16 sera (8.8%) with mannose-binding lectin (MBL) deficiency, 1 serum (0.4%) with C4 deficiency, 1 serum (0.4%) with C9 deficiency, and 1 serum (0.4%) with B deficiency were observed in the HD group, and 18 sera (8.8%) with MBL deficiency and 1 serum (0.5%) with B deficiency were observed in the control group. There were no significant differences in the 5-year mortality rate between each complement-deficient group and the complement-sufficient group among the HD patients.ConclusionThis is the first report that profiles complement deficiencies by simultaneous measurement of functional activities of the three complement pathways in HD patients. Hemodialysis patients frequently suffer from infections or malignancies, but functional complement deficiencies do not confer additional risk of mortality.

A novel measurement method for activation of the lectin complement pathway via both mannose-binding lectin (MBL) and L-ficolin

Mannose-binding lectin (MBL), L-ficolin and H-ficolin are human serum lectins, all of which form complexes with MBL-associated serine proteases (MASP). The lectin-MASP complexes bind to the surface of microbes, leading to activation of the lectin pathway of complement. Enzyme-linked immunosorbent assays (ELISA) of the lectin pathway activity reported so far determined the activity via either MBL or L-ficolin, but an assay of activity via plural host defense lectins has not been established. To measure the lectin pathway activation mediated by plural lectins simultaneously, we developed an ELISA system in which N-acetylglucosamine-pentamer conjugated to dipalmitoylphosphatidylethanolamine (GN5-DPPE) was employed as a ligand for the lectins. In our ELISA system, both purified MBL and L-ficolin isolated from serum diluted in a buffer containing high ionic NaCl bound to GN5-DPPE and activated C4. Purified H-ficolin was not capable of binding to GN5-DPPE. MBL and L-ficolin in MBL-sufficient serum also bound to GN5-DPPE and activated C4. Mannose and N-acetylgalactosamine inhibited binding of MBL and L-ficolin to GN5-DPPE, respectively. MBL-deficient serum that had been depleted of L-ficolin did not exhibit C4 activation, but addition of both or either purified MBL and/or L-ficolin to the serum restored the activation in a dose-dependent manner. Thus, C4 cleaving activity could be evaluated with the co-existence of MBL and L-ficolin in vitro. In conclusion, we propose a novel method using GN5-DPPE for investigating the MBL- and L-ficolin-dependent lectin pathway and anticipate that this method will be useful in innate immunity and clinical research.

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.

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 Spearmans 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.