Morito Endo

Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis

Henoch-Schönlein purpura nephritis (HSPN) is considered a form of systemic vasculitis of the small blood vessels with immune pathogenesis. In this disorder, the complement system is recognized as an important mechanism of glomerular injury. The aim of this study is to determine whether the lectin pathway, a novel pathway of complement activation, is related to the pathogenesis of HSPN. Renal biopsy material from 10 patients with HSPN was studied immunohistochemically and examined for a clinicopathologic correlation. Serum levels of complement components, including mannose-binding lectin (MBL), and plasma levels of complement activation products were also evaluated in these patients and compared with levels in patients with immunoglobulin A (IgA) nephropathy or mesangial proliferative glomerulonephritis (GN) without IgA deposition (non-IgA GN). Glomerular deposition of components of the pathway, MBL and MBL-associated serine protease (MASP-1), as well as C3b/C3c, C5b-9, and C4-binding protein (C4-bp), was detected in 8 of 10 patients. Although no significant correlation was found between glomerular deposition of MBL/MASP-1 and histological or clinical findings, the biopsies on all patients with MBL/MASP-1 deposits were performed within 20 weeks from the onset of disease. Levels of plasma C4d, the activation fragment of C4, and C4-bp, a soluble regulatory protein of the pathway, were greater in patients with HSPN than in those with non-IgA GN. However, there was no difference in serum MBL levels between the three groups of patients (HSPN, IgA nephropathy, and non-IgA GN). These results suggest that complement activation through the lectin pathway was involved at the onset of HSPN, and this mechanism might be important in the disease pathogenesis.

Complement 3 Is Involved in the Synthetic Phenotype and Exaggerated Growth of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) show the synthetic phenotype and exaggerated growth in comparison with VSMCs from normotensive Wistar-Kyoto (WKY) rats. We investigated genes associated with the synthetic phenotype and exaggerated growth of VSMCs from SHR by microarray. Expression of 1300 transcripts was evaluated by microarray with total mRNA extracted from mid-layer aortic smooth muscle of 3-week-old SHR/Izumo and WKY/Izumo rats. mRNAs encoding sodium-dependent neurotransmitter transporter, epidermal growth factor precursor, EEF2, leptin receptor long-isoform b, clathrin assembly protein short form, and preprocomplement 3 (pre-pro-C3) were expressed only in aortic smooth muscle from SHR by microarray and by reverse-transcription polymerase chain reaction analysis. Pre-pro-C3 mRNA was detected only in cultured VSMCs from SHR. Exogenous C3 changed VSMCs to the synthetic phenotype. Antisense oligodeoxynucleotides (ODN) to C3 reduced the higher level of DNA synthesis in VSMCs from SHR. Antisense ODN to C3 increased expression of SM22&agr; mRNA and decreased expression of osteopontin and matrix Gla mRNAs. It also decreased expression of growth factor mRNAs in VSMCs from SHR. In conclusion, we have shown that C3, independent of other complement molecules, has direct effects on the phenotype of VSMCs and stimulates growth of these cells. C3 is produced only by VSMCs from SHR. Therefore, C3 may be the gene underlying the synthetic phenotype and exaggerated growth of VSMCs from SHR. C3 may be a new target for the treatment of hypertension.

Complement 3 activates the renal renin-angiotensin system by induction of epithelial-to-mesenchymal transition of the nephrotubulus in mice

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

Complement Activation Accelerates Glomerular Injury in Diabetic Rats

A known inhibitor of the complement cascade (K-76 COONa) was administered to an inbred diabetic rat model to investigate whether the complement system may play a role in the progression of diabetic glomerulosclerosis. Drinking water containing K-76 COONa was available continuously to inbred diabetic rats (Otsuka Long-Evans Tokushima Fatty, OLETF) from the age of 25 to 55 weeks (Group L). Drinking water without K-76 COONa was similarly available to OLETF rats (Group H) and nondiabetic control rats (Long-Evans Tokushima Otsuka, LETO) (Group C). The levels of plasma glucose (mg/dl) at the 55th week were 156 ± 16 in Group C, 252 ± 18 in Group L and 349 ± 93 in Group H. There was no significant difference in the degree of diabetes between Group L and Group H. The levels of urinary protein at 55 weeks of age (mg/day) were 2.1 ± 0.4 in Group C, 11.6 ± 1.5 in Group L and 18.0 ± 2.8 in Group H. The level of urinary protein was significantly decreased by the administration of K-76 COONa. Histological examination of renal specimens from the sacrificed rats at 55 weeks of age revealed diffuse mesangial expansion in almost all glomeruli in Group H, exudative lesions in 30% of glomeruli in Group H, and only mild mesangial expansion was recognized in Group L. Immunofluorescence study revealed brilliant staining of C3 and immunoglobulins (Ig) in Group H; trace staining of Ig and no staining of C3 were recognized in Group L. The incubation study with guinea pig serum and glomeruli from rats revealed that Ig and complement components also bound to injured glomeruli in vitro. These data indicate that the complement cascade is activated by injured glomeruli and this activation exacerbates diabetic glomerulosclerosis.

Significant Elevations in Serum Mannose-Binding Lectin Levels in Patients with Chronic Renal Failure

Background/Aims: Mannose-binding lectin (MBL), a liver-derived C-type serum lectin, activates the complement cascade through the lectin pathway. Since the complement system contributes to the host defense against infections and mediates inflammatory processes including atherosclerosis, and since chronic renal failure (CRF) patients are prone to the development of infectious complications and cardiovascular disease, we focused on serum MBL levels in CRF patients who were either uremic, or who were receiving hemodialysis treatment. Methods: MBL levels were measured in the sera of subjects with CRF before they began dialysis treatment (pre-HD patients; n = 23) and in the sera of subjects who were receiving maintenance hemodialysis (HD patients; n = 178), by ELISA using polyclonal anti-rabbit IgG and a monoclonal antibody directed against MBL (3E7). Results: Mean levels (± SD) of serum MBL were significantly higher in pre-HD subjects (4.343 ± 2.533 µg/ml, p < 0.05) and in HD subjects (8.897 ± 4.920 µg/ml, p < 0.05), than in healthy controls (1.452 ± 0.692 µg/ml). Levels were also significantly higher in HD subjects than in pre-HD subjects (p < 0.05). Conclusions: Elevated serum MBL levels in patients with CRF might have significantly influence pathologic conditions such as alterations of the immune system and acceleration of atherogenesis.

Complement 3 is involved in changing the phenotype of human glomerular mesangial cells

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN‐γ and TNF‐α. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose‐dependent manner. C3a decreased expression h‐caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 α1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of α‐smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h‐caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases. J. Cell. Physiol. 213: 495–501, 2007.

Serum Mannose-Binding Lectin Levels in Maintenance Hemodialysis Patients: Impact on All-Cause Mortality

Background/Aims: Mannose-binding lectin (MBL) is characteristic of an acute-phase-reacting protein like C-reactive protein (CRP). However, the prognostic value of the serum MBL level has not been examined. The aim of this study was to evaluate whether the serum MBL level can predict all-cause mortality in hemodialysis (HD) patients. Methods: A total of 131 patients without active infection, who had been on maintenance HD for at least 2 years, were included in this study. The serum MBL, high-sensitivity CRP (hs-CRP) level, nutrition markers, and biochemical parameters were measured in June 1999. The cohort was then followed prospectively for 36 months, and clinical data were recorded. Results: The MBL level of the 131 HD patients was 9.054 ± 5.115 µg/ml (mean ± SD). During the follow-up period, 18 patients (9 males and 9 females) died and 113 (64 males and 49 females) survived. The two leading causes of death were cardiovascular events (n = 6, 33.3%) and infection (n = 4, 22.2%). The serum MBL level was significantly lower among the nonsurvivors (6.596 ± 4.990 µg/ml) than among the survivors (9.445 ± 5.046 µg/ml; p < 0.05). There was a significant, although very weak, correlation between the MBL level and albumin level (p < 0.05), but there was no correlationbetween the MBL level and the hs-CRP level. The patients were divided into two groups according to the serum MBL level (<5 and >5 µg/ml). Multivariate analysis of factors predicting all-cause mortality in multivariate logistic regression analysis identified a serum MBL level <5 µg/ml as a variable that independently predicted all-cause mortality (adjusted odds ratio: 7.632; 95% CI: 2.244–25.961; p = 0.0011). Other significant and independent predictors for mortality included the hs-CRP level (every 100 µg/dl increase), hypertension and diabetes mellitus. Conclusions: Our findings suggest that the serum MBL level is a significant predictor of outcome in HD patients. HD patients with a low level of serum MBL should be carefully monitored.

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.

Nephrotic syndrome with portal, splenic and renal vein thrombosis. A case report.

Background: Thromboembolism is known as a major complication of nephrotic syndrome, but only 4 cases of portal vein thrombosis have been reported as a complication of nephrotic syndrome. All of these 4 cases had acute symptoms, and 3 of 4 were in relapsing phase of nephrotic syndrome when thrombi were found. We describe here a case of 51-year-old woman with fresh nephrotic syndrome that was asymptomatically complicated by portal, splenic and renal vein thrombosis. Conclusion: In the presence of fresh nephrotic syndrome of minimal change, asymptomatic and widely distributed, including portal vein, thrombus formation occurred. If the clinical course shows resistance to therapy, we must consider the complication of venous thrombosis. Anticoagulant therapy with heparin and warfarin was effective and all thrombi disappeared without any other complications.

Autoimmune hemolytic anemia occurred prior to evident nephropathy in a patient with chronic hepatitis C virus infection: case report.

BackgroundRenal involvement in patients with chronic hepatitis C virus infection has been suggested to be due to a variety of immunological processes. However, the precise mechanism by which the kidneys are damaged in these patients is still unclear.Case presentationA 66 year old man presented with the sudden onset of autoimmune hemolytic anemia. Concomitant with a worsening of hemolysis, his initially mild proteinuria and hemoglobinuria progressed. On admission, laboratory tests revealed that he was positive for hepatitis C virus in his blood, though his liver function tests were all normal. The patient displayed cryoglobulinemia and hypocomplementemia with cold activation, and exhibited a biological false positive of syphilic test. Renal biopsy specimens showed signs of immune complex type nephropathy with hemosiderin deposition in the tubular epithelial cells.ConclusionsThe renal histological findings in this case are consistent with the deposition of immune complexes and hemolytic products, which might have occurred as a result of the patients underlying autoimmune imbalance, autoimmune hemolytic anemia, and chronic hepatitis C virus infection.