Hiroyuki Inoshita

Significance of glomerular activation of the alternative pathway and lectin pathway in lupus nephritis

The objective of the present study was to elucidate the association between glomerular complement depositions belonging to the alternative (AP) and lectin (LP) pathways, and clinical findings of lupus nephritis (LN). Immunofluorescence (IF) was performed on 17 LN patients using antibodies against factor B, factor H, properdin, mannose-binding lectin (MBL) and L-ficolin. Compared with factor B/factor H negative patients (n = 9), positive patients (n = 8) showed longer duration of LN (p < 0.05) and more severe interstitial fibrosis (p < 0.05). Eleven patients had properdin deposition in glomeruli, and in three of them, with a duration of LN of less than 1 month, factor B was undetectable. Compared with properdin negative patients (n = 6), positive patients (n = 11) showed significantly higher urinary protein excretion (p < 0.01). MBL/L-ficolin positive patients (n = 11) also had significantly higher urinary protein excretion (p < 0.05) compared with negative patients (n = 6). An independent association was found between glomerular deposition of properdin and that of MBL/L-ficolin (p < 0.01) in addition to factor B/factor H. Traces of glomerular activation of AP and LP reflected the clinical status of LN. It appears that glomerular deposition of each complement component, especially properdin, may be an index of the histological activity of LN.

Properdin has an ascendancy over factor H regulation in complement-mediated renal tubular damage

BackgroundUrinary (U)-complement components have been detected in patients with proteinuric renal diseases, and complement activation via the alternative pathway (AP) is believed to play a role in renal tubular damage. The present study aimed to examine the regulation of complement AP activation in patients with renal tubular damage by focusing on the balance between properdin (P) and factor H (fH).MethodsIn the in vivo studies, U concentrations of P, fH and membrane attack complex (MAC) were measured in patients with renal diseases using an enzyme-linked immunosorbent assay (ELISA), and their relationships with the clinical data were evaluated. In the in vitro studies, human proximal tubular epithelial cells (PTECs) were incubated with normal human serum (NHS), P-depleted serum (PDS), purified P and/or fH. Changes in cell morphology and phenotype were assessed by microscopy, real-time polymerase chain reaction (PCR), immunostaining and a cell viability assay.ResultsThe U-P, fH and MAC concentrations were significantly higher in patients with renal disease than in normal controls and correlated with the U-protein and tubular damage markers. Furthermore, multivariate analysis revealed a relationship between P levels and tubular damage markers. There were no significant changes in morphology and mRNA expression in the AP components (P, fH, fB, C3, C5 and C9) after the addition of up to 25% NHS. Dose-dependent depositions of P or fH were observed after the addition of P or fH on PTECs. Depositions of P were not inhibited by fH in a mixture of a fixed concentration of P and a variable concentration of fH, and vice versa. Preincubation with the fixed concentration of P before the addition of NHS or PDS increased the depositions of P, C3 and MAC compared with incubation with intact NHS or intact PDS only; the depositions of C3 and MAC showed a serum-dependent trend. Preincubation with P before NHS addition significantly suppressed cell viability without causing morphological changes.ConclusionsIn the pathogenesis of renal tubular damage, P can directly bind to PTECs and may accelerate AP activation by surpassing fH regulation.

Antiviral innate immunity disturbs podocyte cell function.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis throughout the world. A majority (approx. 60%) of patients with IgAN experience disease exacerbations associated with an acute respiratory or gastrointestinal illness that appears to represent a viral infection. However, the exact mechanism of the disease exacerbation by viral infection is not understood, especially at the cellular and molecular levels. Here we report that glomerular podocytes express the major sensors for double-stranded RNA (dsRNA), a common byproduct of viral replication. In addition to these receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I)-like helicases (RLHs), podocytes express the collateral proteins required to support intracellular signaling. The pathways that mediate responses to dsRNA are fully functional in podocytes. The transcription factor interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-ĸB) are phosphorylated and translocate to the nucleus, and dsRNA increases synthesis of proteins driven by IRF3 (P54, P56 and P60) or NF-ĸB (interleukin 8 and A20). Furthermore, dsRNA suppresses podocyte cell migration, alters the expression of a panel of podocyte essential proteins (nephrin, podocin and CD2-associated protein or CD2AP) and changes transepithelial albumin flux. These effects are dsRNA sensor-specific: TLR3-/- podocytes do not respond to extracellular dsRNA, while intracellular dsRNA has no effect on podocytes bearing a dominant negative form of the major active RLH. These results demonstrate that innate responses to viruses can disturb podocyte cell function in vitro.

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.

Improvement of peritoneal calcification after parathyroidectomy in a peritoneal dialysis patient.

Peritoneal calcification is one of the complications of peritoneal dialysis (PD). It can become serious, leading to severe abdominal pain and even death. Possible mediators of peritoneal calcification in PD patients are assumed to include acetate buffer, overdosage of vitamin D, repeated peritonitis, hypertonic dialysate, calciphylaxis and secondary hyperparathyroidism (SHPT). However, the mechanism and treatment of peritoneal calcification are controversial. Few reports have appeared on improvement of peritoneal calcification after parathyroidectomy (PTX) for SHPT of long duration. We report herein the case of a 48-year-old man on dialysis for 17 years including PD for 14 years. In 1989, he was admitted to hospital because of end-stage renal disease (ESRD), and started treatment with PD. Abdominal computed tomography (CT) first showed peritoneal calcification in August 2002. Peritoneal calcification did not improve despite conventional treatment including discontinuation of PD, control of calcium phosphate product to less than 55 mg2/dl2, removal of the peritoneal catheter and empirical prednisolone (PSL) usage. The intact parathyroid hormone (i-PTH) level was increased over 1,000 pg/ml and extra-osseous calcification occurred. Total PTX was performed in November 2004. Postoperatively, the i-PTH level decreased immediately and calcium phosphate product was maintained in the reference range. Abdominal CT after PTX showed improvement of peritoneal calcification in September 2005. It appeared that PTX could be used to treat patients with persistent peritoneal calcification not responding to conventional treatment. It was postulated that SHPT might play a crucial role in accelerating peritoneal calcification in PD patients.

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.

Clinical and Laboratory Characteristics That Differentiate Hereditary Angioedema in 72 Patients with Angioedema

BACKGROUND Hereditary angioedema (HAE) is a rare but life-threatening condition that results from mutations in C1-inhibitor (C1-INH). Since distinguishing HAE from other causes of angioedema (AE) is a critical problem in emergencies, the objective of the present study was to clarify the differences between HAE and other forms of AE. METHODS Seventy-two patients with AE were enrolled in this study. The medical history and laboratory data of patients with HAE at the first visit were compared to those with other types of AE. RESULTS Subjects included 23 patients with HAE, 33 with mast cell-mediated AE, 5 with drug-induced AE and 11 with idiopathic AE. The average age of HAE onset (19.5 ± 8.0 years old) was significantly lower than in other groups. A family history of AE was noted in 82.6% of HAE patients, which was significantly higher than other groups. Swelling affecting the extremities and gastrointestinal (GI) tract was observed in the majority (60 to 80%) of HAE patients. Life threatening laryngeal edema was observed in 30.4% of HAE patients. In 95.6% of HAE patients serum levels of C4 were less than the lower limit of the normal range. In our subjects, the sensitivity and specificity of low C4 for HAE were 95.6% and 93.8%, respectively. CONCLUSIONS Early onset of AE, positive family history, recurrent AE in the extremities and GI tract, and suffocation are distinctive characteristics of HAE. A low serum level of C4 is a useful marker for making a differential diagnosis of HAE.

Significance of broad distribution of electron-dense deposits in patients with IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is characterized by mesangial cell proliferation and mesangial expansion with mesangial depositions of IgA. We have found that electron-dense deposits (EDD) are often observed in areas other than paramesangial areas in glomeruli. To compare electron microscopic findings with light microscopic findings and clinical data, we examined the biopsies from 178 patients with IgAN. Patients were divided into two groups: group A had only paramesangial deposits and group B had deposits not only in paramesangial areas but also in other areas. All patients examined in this study had EDD in glomerular paramesangial areas. Thirty-six patients were included in group B. Cellular crescent formation in glomeruli and urinary protein in group B were significantly higher than those in group A (P < 0.01). Serum albumin and estimated glomerular filtration rate (eGFR) in group B were significantly lower than those in group A (P < 0.05). Group B showed a significant positive correlation with histological severity, which is defined in the Japanese Clinical Guidelines on IgAN. In patients with broad distribution of EDD, urinary protein was significantly increased (P < 0.05). Detailed observation of EDD distribution has an impact on evaluation of the disease activity of IgAN.

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.