Profiling dendritic cells subsets in renal tissue of patients with crescentic glomerulonephritis.

BACKGROUND: Dendritic cells (DCs) have been speculated to be involved in the pathogenesis of glomerular diseases. However, the numbers and distribution of DC subsets in the kidneys of patients with crescentic glomerulonephritis (CrGN) have not been clearly elucidated. METHODS: A total of 26 patients with biopsy-proven CrGN were enrolled. Indirect immunofluorescence staining was used to quantify DC subsets in renal specimens. Double staining of HLA with CD11C, BDCA2 and CD209 respectively was performed to detect DC subsets. The correlation between DC subsets infiltrated in the kidney and clinical and pathological parameters was investigated. RESULTS: DC subsets were predominantly present in the kidney interstitium, particularly in the peri-glomerular area. The numbers of CD11C+DCs, BDCA2+DCs and CD209+DCs increased in the patients with CrGN and varied among different types of CrGN. Though significant correlation between DC subsets and the percentage of crescents had not been identified, a notable increase in the number of CD11C+DCs were observed with the chronic development of crescents. Furthermore, patients with severe tubulointerstitial injury exhibited significantly more infiltrations of CD11C+DCs, BDCA2+DCs and CD209+DCs. Moreover, the numbers of CD11C+DCs and BDCA2+DCs were found to correlate with the level of serum C3. CONCLUSIONS: Patients with CrGN showed increased kidney infiltration of DC subsets, primarily localized in the renal interstitium and peri-glomerular region. The correlation between DC subsets and fibrosis of crescent and severe tubulointerstitial injury implied a potential involvement of DCs in the development of CrGN.

The protease inhibitor E64d might attenuate the development of experimental anti-glomerular basement membrane disease through regulating the activation of Th1 cells.

BACKGROUND: Cathepsins have been recently identified as a regulator in the activation of Th1 and Th17 cells, which play an important role in the pathogenesis of anti-glomerular basement membrane (GBM) disease. Whether cathepsins contribute to the development of anti-GBM disease through regulating the activation of CD4+ T cell is still unclear. METHODS: Rats with experimental anti-GBM disease was established by immunization with the nephritogenic T cell epitope α3127-148. E64d, a cysteine cathepsin inhibitor, was administered in vitro and vivo to evaluate the effect of cathepsins on regulating the activation of antigen specific T cells and the development of anti-GBM disease. RESULTS: In rats with experimental anti-GBM diseases, E64d treatment not only reduced the levels of proteinuria, serum creatinine and anti-GBM antibody, but also ameliorated the kidney injury with less glomerular IgG deposition, a lower percentage of crescents and less infiltration of CD4+ T cells, CD8+ T cells and macrophages, as well as a lower percentage of splenic Th1 cells. In vitro, E64d treatment could significantly reduce the production of IFN-γ in the supernatant which might be produced by the activation of Th1 cells after being recalled with the autoantigen α3127-148. We also found the CD4+ T cells of rats with anti-GBM disease had an increased expression of cathepsin L (Cts-L), and the percentage of CD4+ T cells with extracellular expression of Cts-L was obviously higher, indicating it as a potential key regulator. CONCLUSIONS: E64d might attenuate the development of anti-GBM disease by participating in the activation of Th1 cells, indicating it as a potential drug for anti-GBM disease in the future.

Significance of thyroid dysfunction in the patients with primary membranous nephropathy.

BACKGROUND: Thyroid dysfunction is common in patients with nephrotic syndrome, especially patients with primary membranous nephropathy (pMN). In view of both MN and thyroid dysfunction are associated with autoimmunity, the current study aimed to elucidate the significance of thyroid dysfunction in patients with pMN. METHODS: Four hundred and twenty patients with biopsy-proven pMN from 2018-2021 were retrospectively enrolled. Clinical and pathological parameters, and treatment response of patients with and without thyroid dysfunction were analyzed. RESULTS: Ninety-one (21.7%) patients with pMN suffered from thyroid dysfunction, among which subclinical hypothyroidism (52.7%) was the main disorder. Compared to patients with normal thyroid function, patients with thyroid dysfunction presented with a higher level of proteinuria, a lower level of serum albumin, a higher level of serum creatinine and more severe tubulointerstitial injury at the time of biopsy. But the positive rate and level of circulating anti-phospholipase A2 receptor (PLA2R) antibody were comparable between these two groups. Though following the similar treatment, the percentage of no response to treatment were significantly higher in the patients with thyroid dysfunction (38.6 vs. 20.0%, P = 0.003). Similar to the urinary protein and the positivity of anti-PLA2R antibody, multivariate COX analysis showed thyroid dysfunction was also identified as an independent risk factor for the failure to remission (HR = 1.91, 95%CI, 1.07-3.40, P = 0.029). CONCLUSION: In conclusion, thyroid dysfunction is common in the patients with pMN and might predict a severe clinical manifestation and a poor clinical outcome, which indicated that the thyroid dysfunction might be involved in the disease progression of pMN.

Complement activation profile of patients with primary focal segmental glomerulosclerosis.

BACKGROUND: Studies on adriamycin mice model suggest complement system is activated and together with IgM contributes to the glomerular injury of primary focal segmental glomerulosclerosis (FSGS). We recently reported primary FSGS patients with IgM and C3 deposition showed unfavorable therapeutic responses and worse renal outcomes. Here we examined the plasma and urinary complement profile of patients with primary FSGS, aiming to investigate the complement participation in FSGS pathogenesis. METHODS: Seventy patients with biopsy-proven primary FSGS were enrolled. The plasma and urinary levels of C3a, C5a, soluble C5b-9, C4d, C1q, MBL, and Bb were determined by commercial ELISA kits. RESULTS: The levels of C3a, C5a and C5b-9 in plasma and urine of FSGS patients were significantly higher than those in normal controls. The plasma and urinary levels of C5b-9 were positively correlated with urinary protein, renal dysfunction and interstitial fibrosis. The plasma C5a levels were positively correlated with the proportion of segmental sclerotic glomeruli. The urinary levels of Bb were elevated, positively correlated with C3a and C5b-9 levels, renal dysfunction, and interstitial fibrosis. The plasma C1q level was significantly decreased, and negatively correlated with urinary protein excretion. Urinary Bb level was a risk factor for no remission (HR = 3.348, 95% CI 1.264-8.870, P = 0.015) and ESRD (HR = 2.323, 95% CI 1.222-4.418, P = 0.010). CONCLUSION: In conclusion, our results identified the systemic activation of complement in human primary FSGS, possibly via the classical and alternative pathway. The activation of complement system was partly associated with the clinical manifestations, kidney pathological damage, and renal outcomes.

Experimental Antiglomerular Basement Membrane GN Induced by a Peptide from Actinomyces.

BACKGROUND: Antiglomerular basement membrane (anti-GBM) disease is associated with HLA-DRB1*1501 (the major predisposing genetic factor in the disease), with α3127-148 as a nephritogenic T and B cell epitope. Although the cause of disease remains unclear, the association of infections with anti-GBM disease has been long suspected. METHODS: To investigate whether microbes might activate autoreactive T and B lymphocytes via molecular mimicry in anti-GBM disease, we used bioinformatic tools, including BLAST, SYFPEITHI, and ABCpred, for peptide searching and epitope prediction. We used sera from patients with anti-GBM disease to assess peptides recognized by antibodies, and immunized WKY rats and a humanized mouse model (HLA-DR15 transgenic mice) with each of the peptide candidates to assess pathogenicity. RESULTS: On the basis of the critical motif, the bioinformatic approach identified 36 microbial peptides that mimic human α3127-148. Circulating antibodies in sera from patients with anti-GBM recognized nine of them. One peptide, B7, derived from Actinomyces species, induced proteinuria, linear IgG deposition on the GBM, and crescent formation when injected into WKY rats. The antibodies to B7 also targeted human and rat α3127-148. B7 induced T cell activation from human α3127-148-immunized rats. T cell responses to B7 were detected in rats immunized by Actinomyces lysate proteins or recombinant proteins. We confirmed B7's pathogenicity in HLA-DR15 transgenic mice that developed kidney injury similar to that observed in α3135-145-immunized mice. CONCLUSIONS: Sera from patients with anti-GBM disease recognized microbial peptides identified through a bioinformatic approach, and a peptide from Actinomyces induced experimental anti-GBM GN by T and B cell crossreactivity. These studies demonstrate that anti-GBM disease may be initiated by immunization with a microbial peptide.

A Modified Peptide Derived from Goodpasture Autoantigen Arrested and Attenuated Kidney Injuries in a Rat Model of Anti-GBM Glomerulonephritis.

BACKGROUND: In Goodpasture disease, the noncollagenous domain 1 of the α3 chain (α3NC1) of type IV collagen is the main target antigen of antibodies against glomerular basement membrane (GBM). We previously identified a nephritogenic epitope, P14 (α3127-148), that could induce crescentic nephritis in WKY rats, and defined its core motif. Designing a modified peptide, replacing critical pathogenic residues with nonpathogenic ones (on the basis of homologous regions in α1NC1 chain of type IV collagen, known to be nonpathogenic), might provide a therapeutic option for anti-GBM GN. METHODS: We synthesized a modified peptide, replacing a single amino acid, and injected it into α3-P14-immunized rats from day 0 (the early-treatment group) or a later-treatment group (from days 17 to 21). A scrambled peptide administrated with the same protocol served as a control. RESULTS: The modified peptide, but not the scrambled peptide, attenuated anti-GBM GN in both treatment groups, and halted further crescent formation even after disease onset. Kidneys from the modified peptide-treated rats exhibited reductions in IgG deposits, complement activation, and infiltration by T cells and macrophages. Treatment also resulted in an anti-inflammatory cytokine profile versus a proinflammatory profile for animals not receiving the modified peptide; it also reduced α3-P14-specific T cell activation, modulated T cell differentiation by decreasing Th17 cells and enhancing the ratio of Treg/Th17 cells, and inhibited binding of α3-P14 to antibodies and MHC II molecules. CONCLUSIONS: A modified peptide involving alteration of a critical motif in a nephritogenic T cell epitope alleviated anti-GBM GN in a rat model. Our findings may provide insights into an immunotherapeutic approach for autoimmune kidney disorders such as Goodpasture disease.

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.

The critical amino acids of a nephritogenic epitope on human Goodpasture autoantigen for binding to HLA-DRB1*1501.

BACKGROUND: Anti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. METHODS: A line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. RESULTS: P14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the ß chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. CONCLUSION: We identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.

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.

Clinical Significance of IgM and C3 Glomerular Deposition in Primary Focal Segmental Glomerulosclerosis.

BACKGROUND AND OBJECTIVES: Glomerular IgM deposition is commonly shown in primary FSGS and sometimes accompanied by C3 deposition. Clinical presentation and treatment outcomes of these patients are not investigated in detail. DESIGN, SETTING, PARTICIPANTS, &MEASUREMENTS: One hundred six consecutive patients with biopsy-proven primary FSGS from 2004 to 2014 were enrolled retrospectively. Clinical features and treatment outcomes were compared between patients with and without IgM/C3 deposition. RESULTS: Fifty-eight (54.7%) patients presented with IgM glomerular deposition on sclerotic segments. C3 and C1q depositions were shown exclusively in patients with IgM deposition (34.5% versus 0.0%; P<0.001 and 8.6% versus 0.0%; P=0.04, respectively). Patients with IgM deposition were younger (median; range: 24.5; 18.8-39.0 versus 46.5; 26.0-64.0 years old; P=0.001), had higher level of serum IgM (142.5; 96.3-206.0 versus 107.0; 71.0-140.0 mg/dl; P=0.01), and had higher level of eGFR (median; range 97.7; 48.0-135.8 versus 62.1; 33.7-93.9 ml/min per 1.73 m(2); P=0.01) at the time of kidney biopsy. The percentage of sclerosis lesions was significantly higher in patients with C3 deposition (median; range: 21.7%; 15.3%-31.1% versus 9.2%; 6.6%-20.0%; P=0.002). Although patients received comparable immunosuppressive treatments during 58.9 (29.5-81.1) months of follow-up, a significantly higher prevalence of refractory cases (no response or steroid dependent) occurred in patients with combined IgM and C3 deposition compared with patients with IgM deposition alone or without IgM deposition (58.8% versus 22.2% versus 15.6%, respectively; P=0.004). Multivariate analysis identified combined IgM and C3 deposition (odds ratio, 11.32; 95% confidence interval, 2.26 to 56.65; P=0.003) as an independent risk factor for refractory patients; 19 of 98 patients developed renal dysfunction when their serum creatinine levels increased >30% from baseline and reached >1.5 mg/dl. Combined IgM and C3 deposition (hazard ratio, 5.67; 95% confidence interval, 1.34 to 23.84; P=0.02) was identified as an independent risk factor for renal dysfunction. CONCLUSIONS: Patients with primary FSGS and IgM and C3 deposition showed unfavorable therapeutic responses and worse renal outcomes, which indicate that IgM and C3 deposition might involve disease progression via complement activation.

Patients With Combined Membranous Nephropathy and Focal Segmental Glomerulosclerosis Have Comparable Clinical and Autoantibody Profiles With Primary Membranous Nephropathy: A Retrospective Observational Study.

Patients with combined membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS) have been reported with different clinical significance. Investigations on the possible mechanisms of the combined glomerular lesions are necessary but scarce. Twenty patients with both MN and FSGS lesions were enrolled in the study. Sixty-five patients with primary MN and 56 patients with primary FSGS were used as disease controls. Clinical data on renal biopsy and during follow-up were collected. Circulating anti-phospholipase A2 receptor (PLA2R) antibody, glomerular PLA2R expression, IgG4 deposition, and soluble urokinase receptor (suPAR) levels were detected. We found that patients with combined lesions presented with older age, less proteinuria, higher albumin, and better renal function on biopsy. These were comparable to the patients with primary MN, but differed from the patients with primary FSGS. Patients with combined lesions showed higher stages of MN, no cellular variant on FSGS classification, and more common (100.0%) tubulointerstitial injury than both primary MN and primary FSGS patients. In the patients with combined lesions, 80.0% had circulating anti-PLA2R antibody and 68.4% had IgG4 predominant deposition in glomeruli, which were comparable to primary MN. The patients with combined lesions had significantly lower urinary suPAR concentrations, than the primary FSGS patients (315.6 ±â€Š151.0 vs 752.1 ±â€Š633.9 pg/µmol; P = 0.002), but similar to the primary MN patients (267.9 ±â€Š147.5 pg/µmol). We conclude that patients with combined MN and FSGS may share the same underlying pathogenesis with primary MN. The FSGS lesion might be secondary to primary MN.