Protective effect of astaxanthin on ANCA-associated vasculitis.

OBJECTIVE: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by inflammation and fibrinoid necrosis of medium and small vessels, and its pathogenesis is closely related to inflammation and oxidative stress. Astaxanthin (ATX) is a carotenoid with anti-inflammatory, antioxidant, and immunomodulatory effects. We hypothesized that ATX could play a role in AAV treatment. This study aimed to investigate whether ATX has a protective effect against AAV and to elucidate its regulatory mechanism. METHODS: In vitro experiments, neutrophils isolated from healthy people were treated with ATX or not and cultured with serum from myeloperoxidase (MPO) -ANCA-positive patients and healthy persons. The levels of IL-6 and TNF-α in neutrophil culture supernatant before and after stimulation were measured. Neutrophil extracellular traps (NETs) and intracellular reactive oxygen species (ROS) in neutrophils were detected after stimulation. In vivo study, experimental autoimmune vasculitis (EAV) rat models were established and then treated with ATX via intragastric administration for 6 consecutive weeks. Urinary erythrocytes, urinary proteins, and serum creatinine were detected and HE staining was performed to assess renal injury in rats. Lung hemorrhage was observed by gross dissection and microscopic Prussian blue staining. The level of serum MPO-ANCA was detected. Serum IL-6, TNF-α, superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in rats were measured to explore the effects of ATX on oxidative stress and inflammation in EAV rats. The deposition of MPO in kidney and lung of rats was detected by immunohistochemistry. RESULTS: ATX significantly inhibited neutrophil secretion of inflammatory factors IL-6 and TNF-α. ATX reduced the elevated levels of ROS in neutrophils stimulated by serum from AAV patients and alleviated the release of NETs. ATX administration was observed to reduce the degree of hematuria, proteinuria, and glomerular crescent formation in EAV rats. The degree of pulmonary hemorrhage was significantly reduced. Besides, the serum levels of IL-6 and TNF-α were attenuated, and antioxidant SOD and GSH-px increased in serum. Pathological results showed that MPO deposition was decreased in lung and kidney tissues after ATX treatment. CONCLUSION: ATX could ameliorate the organ damages in EAV rats. It could serve as a hopeful therapy for AAV by its anti-inflammatory and anti-oxidative feature as a unique nature carotenoid.

Pathogenesis of antimicrobial peptides LL-37 and CpG-ODN in ANCA associated vasculitis.

OBJECTIVES: Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) are a group of systemic autoimmune disorders characterized by necrotizing inflammation of small- to medium-sized blood vessels. The pathogenesis of patients with AAV are still in investigation. In this study, we explored the involvement of LL-37 and nucleic acids in AAV. METHODS: 15 patients with AAV diagnosed according to the Chapel Hill definition between October 2014 and July 2015 in the department of Nephrology of Huangdao, affiliated Hospital of Qingdao University were enrolled. 16 patients with chronic bronchitis (CB) were selected  as disease control group. 15 cases of healthy people from Medical Healthy Center were as healthy control group. Peripheral blood mononuclear cells (PBMCs) were collected from these groups and stimulated by LL-37and (or) two types of CpG-ODN for 7 days. The IFN-α and ANCA in vitro were measured by ELISA. The serum IFN-α, LL-37 and ANCA were measured also. RESULTS: The serum level of IFN-α in AAV group was much higher than that in CB group (692.13 ± 407.28 vs 397.07 ± 211.62 pg/ml, p = 0.019), and that in healthy control group (692.13 ± 407.28 vs 251.54 ± 190.46 pg/ml, p < 0.001). The serum level of LL-37 in AAV group was much higher than that in CB group (101.18 ± 66.59 vs 40.23 ± 13.51 ng/ml, p < 0.001, and that in healthy control group (101.18 ± 66.59 vs 27.80 ± 16.86 ng/ml, p < 0.001). Also the level of IFN-α showed a significant positive relationship with ANCA in AAV group both in serum and in supernatant of cultured PBMCs stimulated by LL-37 and (or) CpG-ODN (r = 0.783, p = 0.001; r = 0.575, p = 0.064; r = 0.649, p = 0.031; r = 0.806, p = 0.003). In patients with AAV, the supernatant levels of IFN-α in cultured PBMCs stimulated by LL-37 and (or) CpG-ODN were higher than that without stimulating factor (p < 0.05). The supernatant level of IFN-α in cultured PBMC stimulated by LL-37 alone was lower than that stimulated by CpGA alone (699.57 ± 476.26 vs 2342.63 ± 2025.11 pg/ml, p = 0.001). But the supernatant level of IFN-α in cultured PBMCs stimulated by LL-37 alone was higher than in that stimulated by CpGB alone (699.57 ± 476.26 vs 153.35 ± 78.08 pg/ml, p < 0.001). The supernatant level of IFN-α in cultured PBMCs stimulated by both LL-37 and CpG-ODN was higher than that stimulated by LL-37 or CpG-ODN alone (2550.57 ± 2217.41 vs 699.57 ± 476.26 pg/ml, p = 0.003; 2550.57 ± 2217.41 vs 153.35 ± 78.08 pg/ml, p = 0.001; 2660.95 ± 391.31 vs 699.57 ± 476.26 pg/ml, p < 0.001; 2660.95 ± 391.31 vs 153.35 ± 78.08 pg/ml, p < 0.001). Either it is stimulated by LL-37 or CpG-ODN or both, the supernatant level of IFN-α in cultured PBMCs in AAV patients was the highest, that in healthy controls was the lowest. Either stimulated by LL-37 or CPG-ODN, or both, the levels of ANCA production in vitro in AAV groups were statistically significantly higher than those in CB group and healthy control group. CONCLUSIONS: There were higher serum levels of LL-37 and IFN-α in patients with AAV. IFN-α could reach a higher level stimulated by LL-37 and nucleic acids both of which are related to infection. Patients with AAV have ANCA-producing B lymphocytes in the circulation even in remission stage. Infections could induce the relapse of AAV.

Significance of serum peptidylarginine deiminase type 4 in ANCA-associated vasculitis.

OBJECTIVE: To investigate the clinical significance of peptidylarginine deiminase type 4 (PAD4) in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). To make a primary observation on the relationship of chronic bronchitis and bronchiectasis (CB) with the pathogenesis of AAV by PAD4. METHODS: The sera from 13 patients with AAV, 13 patients with CB, 11 patients with rheumatoid arthritis (RA), 11 patients with primary chronic kidney disease (CKD) and 12 normal controls were collected. Serum PAD4 was detected using commercial ELISA kits. The serum levels of PAD4 were compared not only among the different groups but also between the activity and remission stage of the same disease. The associations between serum PAD4 and the Birmingham Vasculitis Activity Score (BVAS) of AAV were further investigated. RESULTS: (1) The serum levels of PAD4 in patients with AAV, RA and CB at activity stage were all higher than that in the normal controls (P<0.001, respectively, α'=0.007). The serum level of PAD4 in patients with CB at remission stage and that in CKD group were not found elevated compared with the normal controls (P=0.02, P=0.085, respectively, α'=0.007). (2) At activity stage, among the groups of simple AAV, AAV with a long history of CB and CB without AAV, no significant difference was detected. While at remission stage among the 3 groups, the serum level of PAD4 was at the lowest level in CB group without AAV. (3) The serum level of PAD4 in some patients with CB without AAV were found still higher at remission stage. (4) The serum level of PAD4 in AAV with renal damage at activity stage was positively correlated with BVAS (the activity score of AAV, r=0.71, P=0.02). CONCLUSION: PAD4 is involved in the pathogenesis of AAV. Whether some patients with CB might progress to AAV by the link with PAD4 still need further investigation.

Modified C-reactive protein might be a target autoantigen of TINU syndrome.

BACKGROUND AND OBJECTIVES: The cross-reactive antigen(s) of tubulointerstitial nephritis and uveitis (TINU) syndrome from renal tubulointerstitia and ocular tissue remain unidentified. The authors' recent study demonstrated that the presence of serum IgG autoantibodies against modified C-reactive protein (mCRP) was closely associated with the intensity of tubulointerstitial lesions in lupus nephritis. The study presented here investigates the possible role of IgG autoantibodies against mCRP in patients with TINU syndrome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: mCRP autoantibodies were screened by ELISA with purified human C-reactive protein in 9 patients with TINU syndrome, 11 with drug-associated acute interstitial nephritis, 20 with IgA nephropathy, 19 with minimal change disease, 20 with ANCA-associated vasculitis, 6 with Sjogren's syndrome, and 12 with amyloidosis. mCRP expression was analyzed by immunohistochemistry in renal biopsy specimens from the 9 patients with TINU syndrome and 40 from disease controls. Frozen normal human kidney and iris were used to demonstrate co-localization of human IgG and mCRP from patients with TINU syndrome with laser scanning confocal microscopy. RESULTS: The mCRP autoantibodies were detected in all nine patients with TINU syndrome, significantly higher than that of those with disease controls (P < 0.05). The renal histologic score of mCRP in TINU syndrome was significantly higher than that in disease controls (P < 0.05). The staining of mCRP and human IgG were co-localized in renal and ocular tissues. CONCLUSIONS: It is concluded that mCRP might be a target autoantigen in TINU syndrome.

Renal neutrophils infiltration in antineutrophil cytoplasmic antibodies-negative pauci-immune crescentic glomerulonephritis.

INTRODUCTION: This study aims to investigate the evidence of neutrophil infiltration in renal tissue from patients with antineutrophil cytoplasmic antibodies (ANCA)-negative pauci-immune crescentic glomerulonephritis (CrGN), and a comparison with their ANCA-positive counterparts was performed. METHODS: Renal biopsy specimens from 31 patients with pauci-immune CrGN were collected. Twelve patients were ANCA negative, and 19 patients were ANCA positive. Neutrophil infiltration was investigated by staining of 2 markers, CD15 and myeloperoxidase (MPO), using immunohistochemistry. RESULTS: Positive-stained cells of CD15 (CD15) and MPO (MPO+) were mainly located in glomeruli with cellular crescents and segmental fibrinoid necrotic lesions and in periglomerular area of cellular crescents, especially at the area of ruptured Bowman capsule. Scanty positive staining was found in normal renal tissue. The number of CD15 cells in the glomeruli and periglomerular area was significantly higher in ANCA-negative group than in ANCA-positive group (in glomeruli: 2.70 ± 1.61 versus 1.38 ± 0.85, P = 0.019; in periglomerular area: 4.35 ± 4.36 versus 1.48 ± 1.67, P = 0.047). The number of MPO+ cells in the periglomerular area was also significantly higher in ANCA-negative group than that in ANCA-positive group. CONCLUSIONS: Renal neutrophil infiltration might play a pathogenic role in ANCA-negative pauci-immune CrGN, and the neutrophil infiltration might be more severe in ANCA-negative pauci-immune-CrGN than in ANCA-positive one.

Differential deposition of C4d and MBL in glomeruli of patients with ANCA-negative pauci-immune crescentic glomerulonephritis.

OBJECTIVE: Our previous study suggested involvement of alternative pathway activation of complement in ANCA-positive pauci-immune crescentic glomerulonephritis (CrGN). This study was to investigate the evidence of complement activation in renal biopsy specimens of patients with ANCA-negative pauci-immune CrGN. METHODS: Renal biopsy specimens from 12 patients with ANCA-negative pauci-immune CrGN were used to detect the staining of membrane attack complex (MAC), C3d, C4d, mannose-binding lectin (MBL), factor B by immunohistochemistry, and/or immunofluorescence. Renal tissue from eight patients with minimal change disease (MCD) and renal tissue obtained from a normal kidney and the normal parts of a nephrectomized kidney due to renal carcinoma was used as disease and normal controls, respectively. RESULTS: MAC and C3d could be detected in the active renal lesions of cellular crescents in each of the 12 ANCA-negative CrGN patients but not or scarcely detected in patients with MCD and in normal renal tissue. The deposition of other complement components in the 12 patients was heterogeneous. Although none of them had C1q staining, eight of the 12 were C4d positive and six out of the eight were MBL positive. The remaining four only had evidence of the alternative pathway activation by positive staining of C3d, factor B, and MAC. The staining intensity of C4d and MBL was much higher in dialysis-dependent patients than that in dialysis-independent patients. CONCLUSION: Complement activation was involved in renal damage of human ANCA-negative pauci-immune CrGN but with heterogeneous activation pathways. Positive staining of C4d and MBL might be associated with poor renal outcome.

Complement deposition in renal histopathology of patients with ANCA-associated pauci-immune glomerulonephritis.

BACKGROUND: The pathogenesis of ANCA-associated pauci-immune glomerulonephritis has not been fully elucidated. Several studies had suggested that complement deposition could be detected in renal histopathology. The current study investigated the clinical and pathological significance of complement deposition in renal histopathology of patients with ANCA-associated pauci-immune glomerulonephritis. METHODS: Renal biopsy specimens from 112 patients with ANCA-associated pauci-immune glomerulonephritis were investigated using direct immunofluorescence, light and electron microscopy. For direct immunofluorescence, IgG, IgA, IgM, C3c and C1q staining on fresh frozen renal tissue were routinely performed immediately after a renal biopsy. Complement deposition was defined as the presence of C3c or C1q for at least 1+ in a 0-4+ scale. Clinical and histopathological data between patients with and without complement deposition were compared. RESULTS: In direct immunofluorescence microscopy, C3c and C1q could be detected in glomerular capillary wall and/or mesangium in the specimens of 37/112 (33.0%), 7/112 (6.3%) patients, respectively. Compared with patients without C3c deposition, patients with C3c deposition had a higher level of urinary protein (P < 0.01) and poorer initial renal function (P < 0.05). CONCLUSION: Complement deposition was not rare in renal histopathology of human ANCA-associated pauci-immune glomerulonephritis, which was associated with more severe renal injury.

Complement activation is involved in renal damage in human antineutrophil cytoplasmic autoantibody associated pauci-immune vasculitis.

OBJECTIVE: This study was to investigate the evidence for complement activation in renal biopsy specimens of patients with myeloperoxidase (MPO)-antineutrophil cytoplasmic autoantibody (ANCA)-associated pauci-immune vasculitis. METHODS: Renal biopsy specimens from seven patients with MPO-ANCA positive pauci-immune necrotizing crescentic glomerulonephritis (NCGN) were used to detect the staining of membrane attack complex (MAC), C3d, C4d, mannose-binding lectin (MBL), factor B and factor P using immunohistochemistry and immunofluorescence. Renal tissue from seven patients with minimal change disease (MCD) and two normal renal tissue were used as controls. RESULTS: MAC, C3d, factor B and factor P could be detected in glomeruli and small blood vessels with active vasculitis of patients with pauci-immune AAV, but not or scarcely in patients with MCD and in normal renal tissue. C3d and factor B co-localized with MAC, factor P colocalized with C3d. MBL and C4d were not detected in patients with AAV. CONCLUSION: The alternative pathway of the complement system is involved in renal damage of human pauci-immune AAV.