Monomeric C-reactive protein promotes platelets to release mitochondrial DNA in anti-neutrophil cytoplasmic antibody-associated vasculitis.

Although high level of circulating C-reactive protein (pCRP) is considered as a biomarker for disease activity, the significance of CRP in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has not been clarified. We once reported in AAV, pentameric CRP (pCRP) could dissociate into monomeric CRP (mCRP) and activate platelets. Recent studies have demonstrated that the activated platelets can release mitochondrial DNA (mtDNA). The purpose of this study was to further study the relationship between mCRP and platelets in AAV. We found the plasma level of mCRP in AAV patients was significantly higher than that of normal control and positively correlated with the proportion of mCRP-positive platelets. Platelets isolated from one normal donor could be activated by plasma from 5 AAV patients and this effect could be attenuated when mCRP had been removed. Only 0.1 µg/mL of recombinant mCRP was needed for inducing platelets to release mtDNA via interaction with lipid raft and through p38 MAPK/NF-κB pathway. The mCRP binding on platelets depended on the C-terminal octapeptide (aa 199-206). The released mtDNA did not induce respiratory burst alone, but enhanced the ANCA-induced neutrophils respiratory burst after binding Toll-like receptor 9 (TLR9). The mtDNA released by mCRP-activated platelets also enhanced thrombin generation of plasma. In conclusion, our data demonstrate that mCRP can bind platelets via interaction with lipid raft and induce the release of mtDNA. The released mtDNA can enhance the pathogenicity of ANCA and promote activation of coagulation system in AAV.

Circulating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase chain 6 is associated with disease activity of anti-neutrophil cytoplasmic antibody-associated vasculitis.

BACKGROUND: Increased serum and urinary mitochondrial DNA have been demonstrated in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Here we investigated the significance of serum nicotinamide adenine dinucleotide-ubiquinone oxidoreductase chain 6 (ND6), which is encoded by mtDNA and can attract neutrophils, in AAV. METHODS: Thirty-seven AAV patients (32 patients with positive myeloperoxidase-ANCA and 5 patients with proteinase 3-ANCA) were enrolled. Relationship between serum ND6 and clinico-laboratory characteristics were analyzed. RESULTS: The ND6 level of patients was higher than normal people (46.56 ± 23.67 pg/mL vs. 4.95 ± 2.45 pg/mL, P < 0.001) The ND6 levels of patients who needed hemodialysis at disease onset and who had pulmonary hemorrhage (PH) were higher than that of the corresponding controls (P = 0.004 and 0.044 respectively). The ND6 level negatively correlated with the percentages of normal glomeruli in kidney biopsy. The AUC of ROC curve to diagnose hemodialysis and PH was 0.804 and 0.750 respectively. ND6 level positively correlated with Birmingham Vasculitis Activity Score in active disease, and returned to normal after remission. Patients with higher serum ND6 had higher mortality (P = 0.023). CONCLUSIONS: Serum ND6 increases in active AAV, and its level correlates with the severity of disease. High ND6 level is associated with severe organ injury and predicts poor prognosis of AAV.

Urinary mitochondrial DNA is a useful biomarker for assessing kidney injury of antineutrophil cytoplasmic antibody -associated vasculitis.

BACKGROUND: The value of urinary mitochondrial DNA (mtDNA) for assessing kidney injury of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) was investigated. METHODS: Thirty-nine kidney biopsy-proved myeloperoxidase (MPO)-ANCA associated AAV patients were enrolled and analyzed. RESULTS: The average urinary mtDNA of patients was significantly higher than that of normal controls (3372.74 ± 1859.72 vs. 474.90 ± 123.59 copy/nmol creatinine, p < 0.001). The patients who needed dialysis at disease onset had the highest levels of urinary mtDNA (5072.23 ± 1302.87 copy/nmol creatinine). Urinary mtDNA positively correlated with urinary neutrophil gelatinase-associated lipocalin (R = 0.661, P < 0.001) and negatively correlated with estimated glomerular filtration rate (R = -0.515, P = 0.001). The urinary mtDNA level of crescentic class (4703.08 ± 1744.31 copy/nmol creatinine) was higher than that of mixed class (3258.14 ± 1158.99 copy/nmol creatinine) and focal class (2268.15 ± 1897.63 copy/nmol creatinine). Univariate correlation analysis showed urinary mtDNA positively correlated with interstitial neutrophils (R = 0.471, P = 0.048) and glomerular neutrophils (R = 0.673, P = 0.002) in kidney biopsy. Among 13 patients who needed hemodialysis at disease onset, 10 patients who got renal recovery had higher urinary mtDNA than 3 patients who remained dialysis dependent (5455.20 ± 1174.64 vs. 3795.67 ± 893.34 copy/nmol creatinine, p = 0.047). CONCLUSIONS: Urinary mtDNA increases in AAV with kidney injury, and its levels correlate with the severity of kidney injury and neutrophils infiltration in pathology.

Pathological severity determines the renal recovery for anti-myeloperoxidase antibody-associated vasculitis requiring dialysis at disease onset: a retrospective study.

BACKGROUND: Many patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) need dialysis at disease onset due to severe kidney injury. Determining whether they can become dialysis independent is an important clinical assessment. METHODS: Forty kidney biopsy-proved myeloperoxidase (MPO)-ANCA associated AAV patients who required dialysis at disease onset were enrolled. Relationships between laboratory and pathological characteristics and prognoses were analyzed. RESULTS: Twenty-five patients obtained dialysis independence within 3 months, while the other 15 patients remained dialysis dependent. No sclerotic class was identified among the 40 patients. Only two biopsies exhibited focal class diagnoses and both these patients recovered their renal function. The renal recovery rate of the 20 patients with mixed class was significantly lower than that of the 18 patients with crescentic class (40.0% vs. 83.3%, p = 0.006). Receiver operating characteristics (ROC) curves showed fibrous crescent+global glomerulosclerosis greater than 32.6% was a strong predictor of dialysis dependence with a sensitivity of 93.3% and specificity of 88.0%. When the percentage of fibrous crescent+global glomerulosclerosis exceeded 47.9%, dialysis independence was not possible. Correlation analysis indicated that platelet counts were negatively correlated with the percentage of fibrous crescent+global glomerulosclerosis (R = -0.448, p = 0.004). Most patients with increased platelets (84.62%) obtained renal recovery. Compared with methylprednisolone pulse therapy, plasma exchange accelerated renal recovery (29.4 ± 15.6 vs. 41.4 ± 11.7 days, p = 0.039). CONCLUSIONS: For MPO-ANCA AAV who required dialysis at disease onset, crescentic and mixed classes accounted for the majority of patients in our cohort. The renal outcome of mixed class patients was worse than that of crescentic class. A high proportion of fibrous crescent+global glomerulosclerosis is a predictor of dialysis dependence. Increased platelet count is associated with active and reversible renal lesions.

High serum hepcidin is associated with the occurrence of anemia in anti-myeloperoxidase antibody-associated vasculitis with normal kidney function: a cross-sectional study.

The etiology of anemia in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has not been elucidated. In this cross-sectional study, we tried to investigate the relationship between serum hepcidin and anemia in myeloperoxidase (MPO)-ANCA-AAV. Data of 64 newly diagnosed AAV patients who did not have kidney dysfunction or hemorrhage were analyzed. Serum hepcidin was measured with enzyme linked immunosorbent assay. Twenty-three of 64 patients had anemia. Compared with patients without anemia, patients with anemia had higher Birmingham vasculitis activity score [10 (3, 23) vs. 5 (3, 17), p = 0.020], lower levels of serum iron (5.83 ± 1.63 vs. 9.76 ± 1.54, p < 0.001) and higher levels of ferrtin [358.00 (59.85, 1314.10) vs. 151.05 (43.00, 645.30), p = 0.006]. All 64 patients had increased levels of serum hepcidin compared with normal controls, while patients with anemia had higher serum hepcidin than patients without anemia (85.30 ± 16.92 ng/mL vs. 53.48 ± 13.32 ng/mL, p < 0.001). In the multivariable analysis, the level of hemoglobin correlated with the levels of serum iron (r = 0.344, p = 0.026) and hepcidin (r = - 0.353, p = 0.022). Low level of serum iron was related to high level of serum hepcidin (r = - 0.472, p = 0.001). Immunosuppressive treatment induced rapid decrease of hepcidin and increase of serum iron on the 1st month, while the recovery of hemoglobin was relatively slow. This study indicated that in MPO-AAV without kidney dysfunction or hemorrhage, the existence of anemia is associated with high level of hepcidin which induces low serum iron and the abnormality of iron utilization.

Severe acute interstitial nephritis induced by valsartan: A case report.

RATIONALE: Angiotensin receptor blocker (ARB) can increase serum creatinine or potassium levels in patients with renal insufficiency, renal artery stenosis, heart failure or hypovolemia, but hardly cause severe kidney injury in patients without any risk factors. A case of severe acute interstitial nephritis (AIN) induced by valsartan was reported here. PATIENT CONCERNS: A 62-year-old female with nausea for 1 month and acute deterioration of kidney function for 2 weeks was admitted. She had a history of hypertension for 5 months and had taken valsartan 40 mg daily for 4 months. Although the valsartan had been stopped for 2 weeks, the serum creatinine continuously increased after admission. Kidney biopsy demonstrated the eosinophils infiltration in interstitium. DIAGNOSES: AIN induced by valsartan. INTERVENTIONS: The patient was treated with glucocorticoid. OUTCOMES: The serum creatinine decreased gradually and got back to normal level 5 months later. Then therapy of glucocorticoid was stopped. Renal artery stenosis was excluded by computed tomography angiography (CTA). LESSONS: Although valsartan-induced allergy has been reported previously, AIN was firstly recognized as a severe complication of this drug. We suggest when there is a ARB-associated continuous deterioration of kidney function for patients without renal insufficiency, renal artery stenosis, heart failure or hypovolemia, AIN should be thought of and therapy with glucocorticoid should be considered if necessary.

Clinical and pathologic characteristics of pauci-immune anti-myeloperoxidase antibody associated glomerulonephritis with nephrotic range proteinuria.

BACKGROUND: Heavy proteinuria in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (GN) is usually considered to be associated with immune deposits in renal biopsy. Nephrotic ANCA GN without immune deposits (pauci-immune) is rare and has not been studied specially. In this study characteristics of these patients are to be investigated. METHODS: Clinical and pathological characteristics from 20 kidney biopsy-proven pauci-immune anti-myeloperoxidase antibody-associated GN patients with nephrotic proteinuria were analyzed and were compared with ANCA GN patients without nephrotic proteinuria. RESULTS: Acute kidney injury (AKI) and gross hematuria were much prevalent but extra-renal involvement was less prevalent in pauci-immune ANCA GN with nephrotic proteinuria than in pauci-immune ANCA GN without nephrotic proteinuria. No more severe hypoalbuminemia, hypercoagulability, hyperlipidemia or higher thrombosis incidence were found between two groups. Compared with patients without nephrotic proteinuria, patients with nephrotic proteinuria had more prevalent crescentic category in histopathology. Proteinuria decreased quickly after treatment but much poorer renal prognosis was found in pauci-immune ANCA GN with nephrotic proteinuria. The results of urinary albumin to total protein ratio and urinary protein electrophoresis showed pauci-immune ANCA GN with nephrotic proteinuria had obvious non-selective proteinuria. CONCLUSIONS: Pauci-immune ANCA GN with nephrotic proteinuria do not have more severe hypoalbuminemia, hypercoagulability or hyperlipidemia than patients without nephrotic proteinuria. Non-selective proteinuria might be the reason. However, pauci-immune ANCA GN with nephrotic proteinuria have more prevalent crescentic category in histopathology, higher incidence of AKI, gross hematuria and poorer renal prognosis despite of good sensitivity to therapy of proteinuria.

The Clinical and Immunologic Features of Patients With Combined Anti-GBM Disease and Castleman Disease.

Patients with both anti-glomerular basement membrane (anti-GBM) disease and Castleman disease have been rarely reported. In this study, we report 3 patients with this combination. They had immunologic features similar to patients with classic anti-GBM disease. Sera from the 3 patients recognized the noncollagenous (NC) domain of the α3 chain of type IV collagen (α3(IV)NC1) and its 2 major epitopes, EA and EB. All 4 immunogloblin G (IgG) subclasses against α3(IV)NC1 were detectable, with predominance of IgG1. In one patient with lymph node biopsy specimens available, sporadic plasma cells producing α3(IV)NC1-IgG were found, suggesting a causal relationship between the 2 diseases. One patient, who achieved remission with antibody clearance and normalization of serum creatinine and interleukin 6 concentrations after plasma exchange and 3 cycles of chemotherapy, experienced recurrence of anti-GBM antibodies and an increase in interleukin 6 concentration after chemotherapy discontinuation because of adverse effects, but both returned to normal after another cycle of chemotherapy. This clinical course and the pathologic findings support the hypothesis that the Castleman disease-associated tumor cells are the source of the anti-GBM autoantibodies.

Hypoalbuminaemia in antineutrophil cytoplasmic antibody-associated vasculitis: incidence and significance.

OBJECTIVES: Hypoalbuminaemia has been proved to be a biomarker of poor prognosis in many diseases. The objective of this study was to investigate the significance of hypoalbuminaemia in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: Data of 117 AAV patients were analysed retrospectively. The relationship between hypoalbuminaemia and disease severity were studied. The influence of albumin on the pathogenetic role of ANCA was investigated in vitro. RESULTS: Among all patients, 52 had light hypoalbuminaemia (30g/L<=albumin<35g/L) and 40 had nephrotic hypoalbuminaemia (albumin <30g/L). Patients with hypoalbuminaemia had higher inflammation levels and more severe kidney injury than patients without hypoalbuminaemia, but no significant difference of the urinary protein levels were found between patients with nephrotic and light hypoalbuminaemia. Multivariate analysis showed serum albumin correlated with age (r=-0.566, p=0.018), C-reactive protein (r=-0.521, p=0.032) and haemoglobin (r=0.512, p=0.036). Patients with nephrotic hypoalbuminaemia had higher incidence of infection, end stage renal disease and all cause mortality during treatment than patients with light hypoalbuminaemia or normal serum albumin. In vitro study indicated albumin could inhibit the binding between ANCA and neutrophils in a concentration dependent manner. Albumin also inhibited the ANCA-induced respiratory burst and neutrophil extracellular traps formation. CONCLUSIONS: Serum albumin have an inhibitory effect on the binding between ANCA and its antigen. The incidence of hypoalbuminaemia in AAV with kidney involvement is high but is not caused by heavy proteinuria. Hypoalbuminaemia is correlated with the high inflammation level and poor prognosis of AAV. Therapy targeting hypoalbuminaemia might benefit patients with AAV.

T cell responses to peptides of Goodpasture autoantigen in patients with anti-glomerular basement membrane disease.

AIM: Cell-mediated autoimmunity, especially autoreactive T cells, is crucial in the initiation of anti-glomerular membrane (GBM) disease. Epitopes for T cells on Goodpasture autoantigen are not fully defined. This study investigated T cell epitopes in anti-GBM patients, aiming to identify the epitopes and their clinical significance. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 13 patients with anti-GBM disease. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. PBMC response to each peptide was detected by proliferation assay. Their associations with clinical features were further analyzed. RESULTS: Peripheral blood mononuclear cells proliferative responses to linear peptides on α3(IV)NC1 could be detected in all patients. Five major epitopes were identified as stimulatory in over half of the patients: α3(IV)NC1127-148 (P14) (69.2%), α3(IV)NC1159-178 (77.8%), α3(IV)NC1179-198 (55.6%), α3(IV)NC1189-208 (P19) (75.0%) and α3(IV)NC1141-154 (57.1%). P14 and P19 were highly recognized in patients comparing with healthy controls (69.2% vs. 0.0%, P = 0.011; 75.0% vs. 0.0%, P = 0.021, respectively). CONCLUSION: T cell proliferation to linear epitopes was detected in human anti-GBM disease. α3127-148 was a mutual T and B cell epitope, implying its initial role in epitope spreading process.

Antibodies against linear epitopes on Goodpasture autoantigen in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.

In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr ≤ 133 µmol/L), 70% of patients with moderate renal dysfunction (133 µmol/L < Scr ≤ 600 µmol/L), and 94% of patients with renal failure (Scr > 600 µmol/L) (P = 0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of EA (P4) and EB (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r = 0.764, P = 0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P = 0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.

The susceptible HLA class II alleles and their presenting epitope(s) in Goodpasture's disease.

Goodpasture's disease is closely associated with HLA, particularly DRB1*1501. Other susceptible or protective HLA alleles are not clearly elucidated. The presentation models of epitopes by susceptible HLA alleles are also unclear. We genotyped 140 Chinese patients and 599 controls for four-digit HLA II genes, and extracted the encoding sequences from the IMGT/HLA database. T-cell epitopes of α3(IV)NC1 were predicted and the structures of DR molecule-peptide-T-cell receptor were constructed. We confirmed DRB1*1501 (OR = 4·6, P = 5·7 × 10-28 ) to be a risk allele for Goodpasture's disease. Arginine at position 13 (ARG13) (OR = 4·0, P = 1·0 × 10-17 ) and proline at position 11 (PRO11) (OR = 4·0, P = 2·0 × 10-17 ) on DRß1, encoded by DRB1*1501, were associated with disease susceptibility. α134-148 (HGWISLWKGFSFIMF) was predicted as a T-cell epitope presented by DRB1*1501. Isoleucine137 , tryptophan140 , glycine142 , phenylalanine143 and phenylalanine145 , were presented in peptide-binding pockets 1, 4, 6, 7 and 9 of DR2b, respectively. ARG13 in pocket 4 interacts with tryptophan140 and forms a hydrogen bond. In conclusion, we propose a mechanism for DRB1*1501 susceptibility for Goodpasture's disease through encoding ARG13 and PRO11 on MHC-DRß1 chain and presenting T-cell epitope, α134-148 , with five critical residues.

The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif.

Goodpasture antigen, the non-collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti-glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar-Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3127-148 ) could induce 90.5% (19/21) of WKY rats developing anti-GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3127-148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3127-148 and later on together with intact human α3(IV)NC1. Antibodies towards α3127-148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross-reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3127-148 with substitution of tryptophan136 , isoleucine137 , leucine139 or tryptophan140 lost its nephritogenicity. Human α3127-148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W136 I137 xL139 W140 . These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti-GBM disease.

Myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis developed from ANCA negative renal limited vasculitis: A case report.

RATIONALE: The relationship between antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) and ANCA-negative vasculitis has not been elucidated. PATIENT CONCERNS: A 64-year-old female with edema and proteinuria was admitted. A kidney biopsy indicated focal proliferative nephritis with crescents in 25% of glomeruli. Serum ANCA was negative. Eighteen months later, systemic symptoms emerged and acute kidney injury occurred. Serum ANCA against myeloperoxidase (MPO) turned positive. Repeated kidney biopsy showed more severe lesion than last time. Immunoglobulin (Ig)G was purified from serum obtained before the first kidney biopsy. Weak ANCA which could not be detected in serum was found in IgG. DIAGNOSES: MPO-ANCA-associated AAV developed from ANCA-negative renal-limited AAV. INTERVENTIONS: The patient was treated with glucocorticoid. OUTCOMES: The serum creatinine decreased to 2.17 mg/dL a week later. MPO-ANCA turned negative when re-examined 3 weeks later. No relapse has been observed during follow-up for 6 months. LESSONS: This is the first reported case about the spontaneous transformation from ANCA-negative renal-limited AAV to ANCA-positive systemic vasculitis. There might be a slow process of epitope spreading in the pathogenesis of disease. Physicians should try their best to detect the ANCA in the diagnose and treatment of ANCA-negative AAV.

T cell infiltration is associated with kidney injury in patients with anti-glomerular basement membrane disease.

Cell-mediated autoimmunity, particularly that involving autoreactive T cells, participates in mediating anti-glomerular basement membrane (GBM) disease. However, direct kidney injury mediated by renal infiltrated T cells has not been clearly elucidated in humans. The T cell profile (CD3, CD4, CD8, IL-17, and foxp3) and macrophage (CD68) were examined by immunohistochemistry on renal biopsy tissues from 13 patients with anti-GBM disease. The correlation between cell infiltration and clinical data was also analyzed. We found that the distribution of T cell infiltration was predominant in the peri-glomerular and interstitial areas. CD3+ T cell infiltratrion around the glomeruli with cellular crescent formations was significantly higher than that around the glomeruli with mild mesangial proliferation. CD8+ T cells significantly accumulated around the glomeruli with cellular crescents without IgG deposits compared to those with IgG deposits. The prevalence of infiltrating CD8+ T cells was correlated with the percentage of ruptured Bowman's capsules. In conclusion, cellular immunity may play a crucial role in the inflammatory kidney injury in anti-GBM patients. The periglomerular infiltration of T cells, especially CD8+ T cells, may participate in the pathogenic mechanism of glomerular damage.

Antibodies to α5 chain of collagen IV are pathogenic in Goodpasture's disease.

Autoantibody against glomerular basement membrane (GBM) plays a direct role in the initiation and development of Goodpasture's (GP) disease. The principal autoantigen is the non-collagenous domain 1 (NC1) of α3 chain of collagen IV, with two immunodominant epitopes, EA-α3 and EB-α3. We recently demonstrated that antibodies targeting α5NC1 are bound to kidneys in GP patients, suggesting their pathogenic relevance. In the present study, we sought to assess the pathogenicity of the α5 autoantibody with clinical and animal studies. Herein, we present a special case of GP disease with circulating autoantibody reactive exclusively to the α5NC1 domain. This autoantibody reacted with conformational epitopes within GBM collagen IV hexamer and produced a linear IgG staining on frozen sections of human kidney. The antibody binds to the two regions within α5NC1 domain, EA and EB, and inhibition ELISA indicates that they are targeted by distinct sub-populations of autoantibodies. Sequence analysis highlights five residues that determine specificity of antibody targeting EA and EB epitopes of α5NC1 over homologous regions in α3NC1. Furthermore, immunization with recombinant α5NC1 domain induced crescentic glomerulonephritis and alveolar hemorrhage in Wistar-Kyoto rats. Thus, patient data and animal studies together reveal the pathogenicity of α5 antibodies. Given previously documented cases of GP disease with antibodies selectively targeting α3NC1 domain, our data presents a conundrum of why α3-specific antibodies developing in majority of GP patients, with α5-specific antibodies emerged in isolated cases, the answer for which is critical for understanding of etiology and progression of the GP disease.

Autoantibodies against Linear Epitopes of Myeloperoxidase in Anti-Glomerular Basement Membrane Disease.

BACKGROUND AND OBJECTIVES: Approximately 20%-30% of patients with anti-glomerular basement membrane disease present coexisting anti-myeloperoxidase (MPO) autoantibodies. We previously showed the recognition of a linear fragment of the MPO heavy chain N-terminus ((1)H, MPO279-409) in plasma from most double-positive patients. Herein, we investigated the frequency of autoantibodies against overlapping (1)H-derived linear peptides in plasma from patients with anti-glomerular basement membrane disease. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We synthesized 13 overlapping linear peptides ((1)H-1 to (1)H-13) covering MPO279-409. We retrospectively collected plasma samples from 67 patients with anti-glomerular basement membrane disease from 1996 to 2012, and we screened them for IgG autoantibodies by ELISA using intact human MPO and the overlapping peptides as antigens, and we further investigated the clinical significance. Autoantibody binding to the linear MPO structure was confirmed by Western blotting. RESULTS: We followed up the 67 patients until 2015, with a median follow-up time of 10.0 (2.3-36.0) months, and 56 ESRD events occurred among the 67 patients with follow-up data. Plasma from 23.9% (16) of the patients recognized intact human MPO, whereas 62.7% (42) plasma samples recognized MPO279-409 linear peptides. Of the 13 linear peptides, (1)H-4 (44.8%, 30 patients) and (1)H-12 (40.3%, 27 patients) exhibited the highest recognition frequencies. Patients with autoantibodies against (1)H-11 or (1)H-12 (MPO371-400) were older (46.1±18.8 versus 34.1±16.6 years; P<0.01), had higher serum creatinine upon diagnosis (median 7.8 mg/dl, interquartile range 4.9-12.6 mg/dl versus median 5.4 mg/dl, interquartile range 2.4-7.3 mg/dl; P=0.02), and had a higher probability of progressing to ESRD; however, multivariate Cox regression analysis showed that (1)H-11 or 12 reaction was not an independent risk factor for renal failure (hazard ratio, 1.2; 95% confidence interval, 0.8 to 2.8; P=0.19). CONCLUSIONS: Autoantibodies against linear peptides of MPO can be detected in the majority of patients with anti-glomerular basement membrane disease, and several are associated with disease severity. The potential common pathogenic mechanism between anti-glomerular basement membrane antibodies and anti-MPO autoantibodies in anti-glomerular basement membrane disease requires further investigation.

Identification of critical residues of linear B cell epitope on Goodpasture autoantigen.

BACKGROUND: The autoantigen of anti-glomerular basement membrane (GBM) disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IV)NC1. Our previous study revealed a peptide on α3(IV)NC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150). This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14. METHODS: 16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c) exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001). P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT) was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001) of antibody reaction after each residue replacement. CONCLUSIONS: We defined GFxF (α3142, 143,145) as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.

The alternative pathway of complement activation may be involved in the renal damage of human anti-glomerular basement membrane disease.

Linear deposition of IgG and complement 3 (C3) along glomerular basement membrane (GBM) is generally revealed in the kidneys of human anti-GBM disease. Our recent studies demonstrated the pathogenic role of complement activation in renal damage of this disease. However, the pathways of complement activation were still paradoxical. In this study, renal biopsy tissues from 10 patients with anti-GBM disease were used to investigate the pathways of complement activation by detecting the deposition of various complement components, including C1q, factor B, factor P (properdin), mannose-binding lectin (MBL), C3d, C4d and C5b-9, using immunohistochemistry and immunofluorescence. We found that C1q, factor B, properdin, C3d, C4d and C5b-9 were detected in all the glomeruli of our patients, along GBM with a linear and/or granular staining pattern. Furthermore, C1q, factor B and properdin co-localized well with C5b-9. The properdin also co-localized well with C3d. However, the deposition of MBL was diffusive in mesangium, GBM, Bowman's capsule and within crescents and was not co-localized with C5b-9 but partially co-localized with C4d. The intensity of factor B deposition (3.3 vs. 1.2, P<0.001) and C5b-9 deposition (3.2 vs. 1.6, P<0.001) was significantly stronger in the glomeruli with crescent formation, compared with the glomeruli without crescents. The complement system is overall activated via both the alternative pathway and classical pathway in the kidneys of human anti-GBM disease. The alternative pathway might play an important role in complement activation induced renal damage.

The clinical and immunological features of patients with combined anti-glomerular basement membrane disease and membranous nephropathy.

The association of anti-glomerular basement membrane (GBM) disease, also known as Goodpasture's disease, with membranous nephropathy (MN) has been well documented. However, little is known about the clinical and immunological features of patients with such a combination. This study was designed to investigate the clinical and immunological features of anti-GBM patients with MN and to provide insight into the pathogenesis of this rare entity. Eight patients with combined anti-GBM disease and MN were found to have significantly lower levels of serum creatinine, a significantly lower proportion of oliguria/anuria, and significantly better renal outcomes compared with 30 patients with classical anti-GBM disease. Antibody levels against the EB conformational epitope of anti-α3(IV)NC1 were significantly lower in these patients, as was their levels of anti-α3(IV)NC1 immunoglobulin G1 (IgG1) and IgG3. Serum antibodies against the M-type phospholipase A2 receptor were undetectable in anti-GBM patients with MN but presented in 13 of the 20 patients with primary MN. Thus, patients with combined anti-GBM disease and MN have distinct clinical features and a different immunological profile of MN.