Feng-Mei Wang

The spectrum of renal thrombotic microangiopathy in lupus nephritis

IntroductionAmong various lupus renal vascular changes, thrombotic microangiopathy (TMA) presented with the most severe clinical manifestations and high mortality. The pathogenesis of TMA in systemic lupus erythematosus (SLE) was complicated. The aim of this study was to assess clinical manifestations, laboratory characteristics, pathological features and risk factors for clinical outcomes of lupus nephritis patients co-existing with renal TMA in a large cohort in China.MethodsClinical and renal histopathological data of 148 patients with biopsy-proven lupus nephritis were retrospectively analyzed. Serum complement factor H, A Disintegrin and Metalloprotease with Thrombospondin type I repeats 13 (ADAMTS-13) activity, antiphospholipid antibodies and C4d deposition on renal vessels were further detected and analyzed.ResultsIn the 148 patients with lupus nephritis, 36 patients were diagnosed as co-existing with renal TMA based on pathological diagnosis. Among the 36 TMA patients, their clinical diagnoses of renal TMA were as followings: 2 patients combining with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, 2 patients combining with anti-phospholipid syndrome, 2 patients with malignant hypertension, 1 patient with scleroderma and the other 29 patients presenting with isolated renal TMA. Compared with the non-renal TMA group, patients with renal TMA had significantly higher urine protein (7.09 ± 4.64 vs. 4.75 ± 3.13 g/24h, P = 0.007) and serum creatinine (159, 86 to 215 vs. 81, 68 to 112 μmol/l, P <0.001), higher scores of total activity indices (AI) (P <0.001), endocapillary hypercellularity (P <0.001), subendothelial hyaline deposits (P = 0.003), interstitial inflammation (P = 0.005), glomerular leukocyte infiltration (P = 0.006), total chronicity indices (CI) (P = 0.033), tubular atrophy (P = 0.004) and interstitial fibrosis (P = 0.018). Patients with renal TMA presented with poorer renal outcome (P = 0.005) compared with the non-TMA group. Renal TMA (hazard ratio (HR): 2.772, 95% confidence interval: 1.009 to 7.617, P = 0.048) was an independent risk factor for renal outcome in patients with lupus nephritis. The renal outcome was poorer for those with both C4d deposition and decreased serum complement factor H in the TMA group (P = 0.007).ConclusionsThere were various causes of renal TMA in lupus nephritis. Complement over-activation via both classical and alternative pathways might play an important role in the pathogenesis of renal TMA in lupus nephritis.

Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN.

Serum complement factor H is associated with clinical and pathological activities of patients with lupus nephritis

OBJECTIVE The aim of this study was to investigate serum complement factor H (CFH) and its associations with clinical and pathological features in patients with LN. METHODS Serum CFH was detected in 241 LN patients, 38 active and 11 inactive patients with SLE without clinical evidence of renal involvement and 51 normal controls. Serum CFH autoantibodies and CFH Tyr402His were screened in the 241 LN patients. CFH deposition in kidneys was detected in some patients. RESULTS Serum CFH levels in patients with LN at active phase were significantly lower than in 38 SLE patients or in normal controls. No serum anti-CFH autoantibodies were detected in patients with LN, and there was no significant difference in CFH Tyr402His distribution between patients with LN and normal controls. Glomerular expression of CFH was stronger than in normal controls. Serum CFH levels were mildly negatively associated with SLEDAI scores (r = -0.204, P = 0.001) and positively associated with serum C3 (r = 0.367, P < 0.001) and haemoglobulin levels (r = 0.193, P = 0.003). Patients with LN class III, subclass IV-S and those with thrombotic microangiopathy had the lowest serum CFH. CONCLUSION Serum CFH levels were associated with disease activity of LN.

Plasma complement factor H is associated with disease activity of patients with ANCA-associated vasculitis

IntroductionIncreasing evidences have demonstrated that activation of alternative complement pathway plays an important role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate the association of complement factor H (CFH), a key regulator of the alternative complement pathway, with the disease activity of AAV.MethodsPlasma CFH levels were measured in 82 patients with myeloperoxidase (MPO)-AAV in active stage. Of the 82 patients, plasma CFH levels of 27 patients were longitudinally measured. Serum anti-CFH autoantibodies were screened in AAV patients. Circulating complement activation profiles including C4d, Bb, C3a, C5a and soluble C5b-9 of AAV patients in active stage were further detected. Associations between plasma CFH levels and clinicopathological parameters as well as the prognosis were analyzed.ResultsPlasma CFH levels were significantly lower in active AAV patients compared with AAV patients in remission and normal controls. Correlation analysis showed that plasma CFH levels inversely correlated with initial serum creatinine, Birmingham Vasculitis Activity Score (BVAS), proportion of total crescents and cellular crescents in renal specimens, and circulating levels of C3a, C5a and Sc5b-9, meanwhile positively correlated with estimated glomerular filtration rate (eGFR), hemoglobin levels and circulating levels of C3. Moreover, multivariate survival analysis revealed that plasma CFH levels were independently associated with composite outcome of death or end stage renal disease (ESRD) in AAV patients, after adjusting for age, gender, hemoglobin level and urinary protein (P = 0.03, HR 0.85, 95 % CI 0.73–0.98) or adjusting for age, gender, total crescents (%) and urinary protein (P = 0.03, HR 0.85, 95 % CI 0.73–0.98), while not as an independent predictor after adjusting for age, gender, serum creatinine and urinary protein (P = 0.57, HR 0.96, 95 % CI 0.83–1.11).ConclusionIn conclusion, plasma CFH levels are associated with disease activity, and, to some extent, associated with composite outcomes of patients with MPO-ANCA-associated vasculitis.

Overactivation of Complement Alternative Pathway in Postpartum Atypical Hemolytic Uremic Syndrome Patients with Renal Involvement.

Postpartum atypical hemolytic uremic syndrome (aHUS) is a life‐threatening syndrome with unclear pathogenesis. The current study aimed to investigate the clinical and pathological features, complement activation status, and the genetic variations in a Chinese cohort of patients with renal biopsy‐proven postpartum aHUS.

Complement Alternative Pathway׳s Activation in Patients With Lupus Nephritis ☆ ☆☆

Objective: The aim of this study was to detect the spectrum of complement activation pathways in circulation and to assess their correlations with clinical and pathologic features in a large lupus nephritis cohort from China. Materials and Methods: Plasma levels of C1q, mannose‐binding lectin, C4d, Bb, C3, C3a, C5a and soluble C5b‐9 were detected by enzyme‐linked immunosorbent assay in 222 patients with active biopsy‐proven lupus nephritis, 34 patients with lupus nephritis at remission, 82 patients with active systemic lupus erythematosus without renal involvement and 39 normal controls. The correlations between levels of complement components and clinicopathological features of these patients were further analyzed. Results: Plasma levels of C1q and C3 significantly decreased, and the levels of Bb, C3a, C5a and soluble C5b‐9 were significantly elevated in patients with active lupus nephritis compared with those in remission, active systemic lupus erythematosus without renal involvement group and normal controls. In the lupus nephritis group, soluble C5b‐9 levels were inversely correlated with C1q and C4d levels (r = −0.412, P < 0.001 and r = −0.221, P = 0.002, respectively), but more strongly correlated with the level of Bb (r = 0.546, P < 0.001). C3b, Bb and C5b‐9 could colocalize on glomeruli in lupus nephritis. Plasma Bb level was significantly correlated with some renal disease activity indices and was a risk factor for renal outcomes (hazard ratio = 1.745; 95% CI: 1.106‐2.754; P = 0.017) in the lupus nephritis group. Conclusions: Our findings suggested that the activation of the complement alternative pathway might play a more important role in the pathogenesis of lupus nephritis, and factor Bb might be a useful marker for evaluating renal disease activity and outcomes.

A method of purifying intact complement factor H from human plasma.

The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacryl S-300 High Resolution HiPrep 16/60 column. All the above steps were performed at 4°C by Fast Protein Liquid Chromatography (FPLC) AKTA Purifier 10 with Frac-900. Identification of the purified CFH was confirmed by SDS-PAGE and Western blot. The following functions of the purified CFH were further analyzed compared with the commercial CFH in vitro: (1) binding ability with C3b; (2) binding ability with mCRP; (3) the protecting function of the hemolysis of sheep red blood cells; (4) the cofactor role for complement factor I-mediated proteolytic inactivation of C3b. Homogeneous CFH was purified from the plasma fraction through the above four steps. The purity and the functions of the purified CFH were comparable to the commercial CFH. The yield of CFH was 26±3% in our study. Compared with previous methods, our method was high yield with high purity. We established a stable and feasible system for purifying intact CFH, which could be used in the lab and clinical investigations.

The dysfunctions of complement factor H in lupus nephritis.

Objective Our previous study showed that plasma levels of factor H (FH) were significantly decreased in patients with lupus nephritis and reflected lupus nephritis activity. The aim of this study was to further investigate in vitro biofunctions of plasma FH in patients with lupus nephritis. Methods FH was purified from the first run of plasma exchange in four active lupus nephritis patients and two non-renal involvement systemic lupus erythematosus (SLE) patients, and plasma from two healthy controls. Then, the biofunctions of the purified FH were analyzed. In addition, FH exons sequencing analysis was performed. Results Homogeneous FH was purified from the plasma fractions and the purity of the purified FH was comparable to the commercial FH. The abilities of FH binding with C3b and mCRP, and its protecting abilities from the lysis of sheep erythrocytes, from No. 3 and No. 4 lupus nephritis patients, decreased significantly compared with those in normal controls. The purified FH from lupus nephritis patients Nos. 2–4 could not induce the phagocytosis of late apoptotic cells significantly compared with normal controls. All four lupus nephritis patients had the known SNP rs1061147 (SCR5, A307A), rs1061170 (SCR7, Y402H), CM050194 (SCR20, S1191W) and CM010322 (SCR20, V1197A), which might be associated with the above dysfunctions. Conclusions Dysfunctions of FH, including the regulations of complement alternative pathway and the clearance of apoptotic cells, were found in some active lupus nephritis patients, which were associated with their clinical phenotypes. The FH SNPs might contribute to the dysfunctions of FH in patients with lupus nephritis.

The functional activities of complement factor H are impaired in patients with ANCA-positive vasculitis

Increasing evidences have demonstrated that the activation of the alternative complement pathway is crucial for the pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Our recent study found that circulating levels of complement factor H (FH), a key regulator of the alternative pathway, were associated with disease activity. In the current study, functional activities of FH were assessed to further explore the potential role of FH in the pathogenesis of AAV. We found that the two patients with ANCA-negative pauci-immune necrotizing crescentic glomerulonephritis exhibited relatively normal functional activities of FH. However, patients with ANCA-positive vasculitis exhibited deficient functional activities of FH, in terms of interaction with and the regulation of C3b, binding to mCRP and endothelial cells, and the protection of host cells against complement attack. Our findings indicate that functional activities of FH are deficient in patients with ANCA-positive vasculitis, potentially contributing to the disease development.

Myeloperoxidase influences the complement regulatory activity of complement factor H

Objective The interaction between neutrophils and activation of alternative complement pathway plays a critical role in the pathogenesis of ANCA-associated vasculitis (AAV). MPO, which can be released from ANCA-stimulated neutrophils, was recently demonstrated to be capable of activating the alternative complement pathway. Here we aimed to investigate the interaction between MPO and factor H (FH), a key regulator of the alternative pathway, and its effect on the functional activities of FH. Methods Detection of FH and MPO on neutrophil extracellular traps (NETs) induced by serum from AAV patients and in kidney biopsies of AAV patients was performed by immunostaining. In vitro binding between MPO and FH was examined by ELISA and surface plasmon resonance. The influence of MPO on the complement regulatory activity of FH was further assessed. Results FH deposited and co-localized with MPO in NETs. In kidney biopsies from AAV patients, MPO was closely adjacent to FH in glomerular capillaries. We demonstrated that MPO binds to FH with an apparent nanomolar affinity and identified short consensus repeats 1-4 of FH as the major binding sites. In terms of functional analysis, MPO inhibited the interaction between FH and C3b and the decay-accelerating activity of FH. The fluid phase and surface cofactor activities of FH upon C3b inactivation were inhibited by MPO. Conclusion Our findings indicate that MPO binds to FH and influences the complement regulatory activity of FH. MPO-FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway.