Membranous nephropathy: a radioimmunologic search for anti-renal tubular epithelial antibodies and circulating immune complexes.

In an effort to elucidate immunopathogenic of membranous nephropathy (MN), freshly collected sera from patients with biopsy proven MN were assayed of circulating immune complexes (ICs) by the Raji cell method and for anti-renal tubular epithelial (RTE) antibodies by a newly established radioimmunoassay (RIA) and by indirect immunofluorescence. 6 of 26 MN patients tested by the Raji cell assay had detectable circulating ICs. However, 5 of these 6 patients had other medical conditions which might also explain the IC reactivity. 29 MN patients and 11 patients with other glomerular diseases had no demonstrable circulating anti-RTE antibodies. This study suggests that if RTE antigens possess a nephritogenic potential for man it is probably only rarely expressed. The inconstant detection of circulating immune complexes in idiopathic MN raises an speculation as to their immunopathogenic significance.     

Expression of complement 3 receptors (CR1 and CR3) on neutrophils and erythrocytes in patients with IgA nephropathy

Expression of C3 receptors (CR1 and CR3) on neutrophils (PMN) was measured in 26 patients with IgA nephropathy (IgA N), 17 normal persons, and 8 patients with non-IgA glomerulonephritis (non-IgA GN) by fluorescence activated cell sorting after labeling with monoclonal antibodies. Mean channel fluorescence for both CR1 and CR3 on PMN was significantly higher in IgA N patients than in either control group (p less than 0.01). (CR1 mean +/- SD 42.5 +/- 10.4 in IgA N, 31.7 +/- 11.5 in normal controls, 27.8 +/- 9.0 in non-IgA GN; CR3 94.0 +/- 16.5, 75.0 +/- 16.6 and 76.7 +/- 15.6, respectively.) No differences were found between the two control groups or between IgA N patients with normal and impaired renal function. These results imply that PMN are activated in IgA N patients. The expression of CR1 and CR3 of PMN may be upregulated by immune complexes (ICs), enhancing both phagocytosis of C3b- or iC3b-coated particles, and absorptive pinocytosis of soluble ICs containing C3b or iC3b. Erythrocyte CR1 and CR3 expression was measured by ELISA and found to be slightly but significantly lower in IgA N patients than in the other 2 groups (CR1 85.3 +/- 28.4 in IgA N, 113.1 +/- 28.8 in normal controls, 109.4 +/- 28.2 in non-IgA GN, p less than 0.02; CR3 80.9 +/- 20.0, 100.4 +/- 19.9, 101.2 +/- 24.9, respectively, p less than 0.05).     

Glomerulonephritis and hereditary angioedema: report of 2 cases

Hereditary deficiency of C1 esterase inhibitor (C1 INH) responsible for hereditary angioedema (HAE) is the most common hereditary complement deficiency. HAE has occasionally been reported in association with lupus erythematosus and with glomerulonephritis (GN). We report 2 cases of GN-associated C1 INH deficiency. Renal manifestations have been discovered respectively 6 to 17 years before onset of attacks. Kidney biopsy of the 1st patient showed diffuse proliferative GN with a rare and scattered wire loop pattern whereas the 2nd patient displayed a type I membranoproliferative GN. Chronic renal failure appeared in both cases and the 2nd patient recently received a kidney transplant. The onset of GN in patients with HAE outline the relationship between a genetic deficiency of complement components, the susceptibility to immune complex (IC) disease and the role of complement and its receptors in the elimination of IC.     

Diagnostic significance and antigen specificity of antineutrophil cytoplasmic antibodies in renal diseases. A prospective multicentre study

In a prospective multicentre study on the clinical significanceof ANCA in renal diseases, sera from 920 patients with rapidlyprogressive renal failure and/or renal disease in associationwith extrarenal signs suggestive of a systemic vasculitis weretested for the presence of ANCA by indirect immunofluorescence(IIF) and ELISA. 193 of 920 cases (20.9%) were positive by IIFand 180 (19.5%) by ELISA, using a ‘crude’ cytoplasmicextract as substrate. The sensitivity and specificity of IIFfor ‘pauci-immune’ crescentic necrotizing GN (CNGN),in association or not with systemic vasculitis, was 87.5 and95.6% respectively. The IIF pattern and antigen specificity (alpha granules andMPO) correlated well with the clinical features: a cANCA pattern(alpha granules) was associated with ENT involvement (probableWegener's granulomatosis); a pANCA pattern (MPO) with ‘idiopathic’CNGN and small-vessel vasculitis without respiratory tract disease(microscopic polyarteritis); patients with a pulmonary-renalsyndrome had either c or pANCA in a similar proportion. Our study confirms a high sensitivity and specificity of ANCAfor patients with CNGN. ANCA should be considered an important diagnostic test in patientswith renal diseases, especially in the presence of rapidly progressiverenal failure.     

Immunodiagnostic aspects of autoantibodies against myeloperoxidase.

The antigen specificity of autoantibodies causing perinuclear staining of granulocytes and monocytes (pANCA) was evaluated by analyzing 3000 sera, which were sent to us for screening of anticytoplasmic antibodies (ACPA, synonym: cANCA, anti-proteinase 3). In 620 sera, perinuclear staining was found. Antigen specificity was investigated by a myeloperoxidase ELISA and indirect immunofluorescence with Hep2 cells specific for antinuclear antibodies (ANA). Only 9.8% of the 620 sera showed reactivity with myeloperoxidase (AMPO), while 85.6% contained ANA which induced a pANCA-like staining. A further 4.6% of the 620 sera were neither ANA nor AMPO positive. Therefore, pANCA in general is only an indication that one should look for AMPO or other antilysosomal autoantibodies, when ANA have been excluded. To investigate the disease specificity of AMPO, we examined sera from patients with several well-defined autoimmune diseases. There were only very few positive results in collagen vascular diseases (3/114) (positive/total), primary systemic vasculitis (1/116) and clinically and histologically proven Wegener's granulomatosis (2/213). On the other hand, AMPO were present in patients with different forms of glomerulonephritis (45/192), especially crescentic glomerulonephritis (CGN) (34/79) without immune deposits in their biopsy specimen (3/30 showed trace deposits of IgM). There were, however, additionally 11 patients with symptoms resembling WG (who were cANCA negative, w/o characteristic WG biopsy), who had no obvious renal symptoms. These findings indicate that AMPO are primarily associated with idiopathic GN, especially CGN. Together with anti-proteinase-3 antibodies and anti-glomerular basement membrane antibodies they are an essential serologic parameter in the diagnosis of unclear systemic diseases with renal involvement.     

Soluble immune complexes in sera of patients with nephritis.

Binding of radioactively labeled C1q was used to detect soluble antigen-antibody complexes in sera collected at the time of renal biopsy from 104 patients with immunofluorescent findings consistent with immune-complex disease. In comparison with data obtained with sera from 85 healthy donors, significantly elevated C1q binding activity was demonstrated in sera from 22 patients. C1q binding was elevated in all four patients whose dominant histologic finding on bright field microscopy was an intense interstitial mononuclear cell infiltrate. High C1q binding activity was found preferentially in sera from patients who had diffuse rather than focal histologic abnormalities by light microscopy, heavy glomerular deposits of C4 and C3 by immunofluorescence and elevated serum creatinine concentrations. However, there were many patients with similar immunofluorescent and bright field microscopic changes in whom circulating complexes were not detected and there was no correlation between the pattern of glomerular localization of immune complexes and the C1q binding activity of the sera. Serial measurements of C1q binding activity in the sera from three patients over a 90-day interval emphasized that immune complexes may be demonstrated by this technique only intermittently in the sera of some patients with renal biopsy evidence of immune-complex disease. Nevertheless, these observations suggest that the C1q binding test may be a useful tool to monitor disease activity in patients with immunologically mediated renal disease.     

Crescentic glomerulonephritis associated with infective endocarditis: renal recovery after immediate surgical intervention

A 57-year-old man was referred to our hospital because of acute cardiac failure and acute renal insufficiency. Laboratory data showed elevation of serum immune complex levels and antineutrophil cytoplasmic antibody (ANCA) titers, with cytoplasmic pattern (C-ANCA) on indirect immunofluorescence (IIF), and proteinase 3 specificity (PR3-ANCA) on solid-phase enzyme-linked immunosorbent assay (ELISA). Hemodialysis therapy was initiated, and this relieved the symptoms of cardiac failure. Echocardiography revealed three-grade aortic insufficiency and two large floating vegetations on the aortic valve. Considering the risk of embolism, we immediately performed aortic valve replacement and surgically removed the vegetations, subsequently giving antibiotic therapy. Six weeks after the operation, the patient's renal function showed marked improvement and the serological abnormalities, except for ANCA titers, had normalized, resulting in no need for dialysis. A renal biopsy specimen revealed diffuse proliferative glomerulonephritis (GN) with crescents including more than 50% of glomeruli, and granular deposits of IgM, C3, and C1q on immunofluorescence. ANCA titers remained high, but the patient's renal function has been stable, indicating a discrepancy between ANCA titers and his clinical course. In this patient, treatment by immediate surgical intervention, performed during the acute phase with active GN and highly reduced renal function, led to dramatic renal recovery. This case suggests that surgical removal of vegetations in the early stage of crescentic GN may result in a good renal outcome in patients with rapidly progressive GN associated with endocarditis. Although it has been suggested that ANCA may have some relationship to GN in endocarditis, in this patient, its pathogenetic significance is questionable. Received: March 10, 2000 / Accepted: May 23, 2000     

Interleukin-1 production by monocytes from patients with glomerulonephritis after stimulation in vitro with soluble immune complexes.

Monocytes from 30 patients with glomerulonephritis (GN) were stimulated with soluble immune complexes (IC). In order to neutralize the effect of prostaglandins, some cultures were incubated in the presence of indomethacin. Interleukin-1 (IL-1) activity was assessed by the comitogenic activity of the crude monocyte supernatants on phytohemagglutinin (PHA)-stimulated murine thymocytes. Our results demonstrate that, upon stimulation with the soluble IC monocytes from GN patients possess an enhanced capacity to produce IL-1, and the levels of IL-1 correlate with disease activity only in one case of acute poststreptococcal GN (AGN), not in all other patients. This enhanced production of IL-1 may contribute to the disordered immunoregulation in GN.     

Clinicopathological study of originally non-lupus “full-house” nephropathy

Background: Glomerular “full-house” immunofluorescence staining commonly indicates lupus nephritis. However, some non-lupus nephropathy also can present with a “full-house” immunofluorescence pattern mimicking lupus nephritis. The goal of this study is to define the clinicopathological spectrum of originally non-lupus “full-house” nephropathy. Methods: Records of 24 patients with “full-house” nephropathy in the absence of clinical or serological evidence of systemic lupus erythematosus (SLE) at the time of renal biopsy were abstracted for demographics, clinical presentation, laboratory data, renal biopsy findings, and clinical follow-up. Results: The clinicopathological diagnoses included membranous glomerulonephritis (GN) (46%), IgA nephropathy (21%), membranoproliferative GN (12.5%), postinfectious GN (12.5%), C1q nephropathy (4%), and unclassified mesangial GN (4%). No one had endothelial tubuloreticular inclusions. One patient originally diagnosed as IgA nephropathy developed anti-DNA antibody and another one patient with membranous GN developed hypocomplementemia 8 months and 10 months after renal biopsy, respectively. The two patients also developed clinical symptoms of lupus subsequently. Conclusions: There was a broad spectrum of glomerular histological findings in non-lupus “full-house” nephropathy. The possibility of “full-house” nephropathy preceding the emergence of overt systemic lupus erythematosus remained to be elucidated.     

Circulating immune complexes in regularly dialyzed patients with chronic renal failure

The prevalence of circulating immune complexes (CIC) was investigated using the C1q binding assay (C1q BA) and the conglutinin binding assay (Kg BA) in 200 patients undergoing maintenance hemodialysis. Increased C1q binding was found in 45% (87 of 194) of the patients, and the modified Kg BA gave elevated values in 31% (20 of 65). The prevalence of CIC was similar in American and Swiss patients, and in patients undergoing hemodialysis, self-dialysis or peritoneal dialysis. In patients with 'nonimmunological' renal diseases, CIC were detected with similar frequency. No change in CIC was noted during hemodialysis in 6 additional patients tested. The abnormality was not related to age, sex, duration of dialysis, hepatitis B antigenemia, bacterial infections, or transfusions. Anti-DNA antibodies were absent in all subjects tested and the results of the C1q BA were not changed by DNase digestion of eight sera with high C1q binding. Rheumatoid factor activity (RF) was detected in approximately one-fifth of the patients, and there was a direct correlation between positive C1q binding and RF. There was no correlation between CIC and lymphocytotoxic antibodies. This study demonstrated a high prevalence of CIC in dialyzed uremic patients and established its relationship to other immunological abnormalities.     

An evaluation of nephelometry and complement-based tests for the detection of circulating immune complexes

Sera from 20 normal adult control subjects and 28 patients suffering from various diseases which may be associated with an immune complex disorder were investigated, using three different techniques for detection of circulating immune complexes (CICs). The sera from the patients were assigned expected positive or negative ratings by the clinicians according to clinical and laboratory criteria. This information as well as the diagnoses was withheld until the results of immune complex determinations were available. The three tests used to detect CICs were laser nephelometry (LN), 125I-C1q binding and measurement of the C3 breakdown product C3c. Serum levels of the complement components C3 and C4 were assessed on the serum specimens from the patients. Results obtained from normal control sera showed that 18 of the 20 and all 20 were negative with the C1q binding technique and LN respectively. Of 16 sera for which a positive result was expected, 5 (31,3%) and 14 (87%) were positive when examined by the C1q binding technique and LN respectively; C3c determination produced no positive results. No false-negative results were obtained with the C1q binding and C3c tests, but 2 out of 16 (13%) results obtained with the LN test were false negative. LN is a rapid, sensitive test for the detection of CICs.     

Comparison Between Total IgG,C1q,and C3d Single Antigen Bead Assays in Detecting Class I Complement-Binding Anti-HLA Antibodies

Background

Complement-binding donor-specific antibodies (DSAs) are associated with antibody-mediated rejection and allograft loss. Novel single antigen bead (SAB) assays—that is, complement component 1q (C1q) and complement component 3d (C3d) assays—have been developed to specifically detect complement-binding DSA, but it remains unclear whether these assays have an improved ability to detect complement-binding DSA as compared with using the total IgG SAB assay with a high mean fluorescence intensity (MFI) cutoff. The aim of this study was to compare the ability of the total IgG, C1q, and C3d SAB assays in detecting complement-binding anti-HLA antibodies.

Limitations of immune complex measurements in colorectal disease

Three techniques (Clq, Raji and L1210 binding assays) alleged to measure circulating immune complexes (ICs) were applied to the sera of 101 patients with colorectal disease (54 carcinoma; 23 inflammatory; 13 benign tumour and 11 miscellaneous) at the time of diagnostic or definitive surgery, and 58 healthy adult controls. Elevated levels in the pathological sera were observed by all 3 methods in order of sensitivity: Raji greater than Clq greater than L1210. However, none of them differentiated between benign, inflammatory and neoplastic conditions nor, in the case of colorectal carcinoma, was there any correlation with stage of disease. With the exception of Raji v. L1210 (r = 0.43, P less than 0.001), correlations between the various assays were poor and levels of serum carcinoembryonic antigen (CEA) did not correlate with ICs measured by any of the techniques. Indeed, the IC assays were even less discriminatory than CEA, which was elevated mainly in the serum of carcinoma patients and which was positively correlated with serum gamma-glutamyl transpeptidase (gamma GT) (r = 0.42, P less than 0.005). The data suggest that the lack of concordance between the IC assays is a reflection of heterogeneity among ICs, interfering factors present in pathological sera, or both. Thus the IC assays deployed here have neither diagnostic nor prognostic utility in colorectal disease at this time, and immunochemical characterization of the serum reactive material detected by the different assays is required.     

Clinical significance of circulating immune complexes detection in chronic glomerulonephritis.

Sera from 168 patients with various types of chronic glomerulonephritis (GN) were assayed for immune complexes (IC) by two independent methods, a modification of the PEG precipitation test (PEG) and the inhibition of complement-dependent lymphocyte rosette formation (RI). Both assays had different reactivities, the RI being more sensitive than the PEG test. Higher percentages of positivity of these tests were observed in the GN groups than in normal controls. However serial measurements demonstrated that IC were present only intermittently in most instances. The presence of IC correlated with disease activity in patients with focal glomerulosclerosis membranous GN and membranoproliferative GN, while in lupus erythematosus it reflected the effects of different treatment regimens although it had no relationship with clinical symptoms. Nevertheless, our results suggest that these tests are of little help to the clinician to appreciate disease activity and monitor therapy in individual GN patients.     

Mesangial deposition of IgA2 subclass and lectin pathway-mediated complement activation in IgA glomerulonephritis

Three pathways are recognized in the complement activation cascade. The aim of our study was to elucidate immunohistologically which complement pathway is associated with the activation in IgA glomerulonephritis (GN) and the relation of IgA subclass to the complement activation. Immunohistological staining was performed on biopsied renal specimens from 36 patients with IgA GN, 10 with systemic lupus erythematosus (SLE) and 16 with other glomerulonephritides using polyclonal antibodies of IgG, IgA, IgM, C3c, C4, C1q and monoclonal antibodies of IgA1, IgA2, mannose-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1). Mesangial deposits of IgA1, IgA2, C3c, C4, MBL and MASP-1 were detected in 19 of the 36 patients with IgA GN, and IgA2 and MBL/MASP-1 were colocalized in the mesangium in these 19 patients. The remaining 17 patients showed mesangial deposition of IgA1 alone. Twelve of these 17 patients presented mesangial deposition of C3c without deposition of C4, MBL and MASP-1. No deposition of C1q was evident in IgA GN patients. Three of the 10 SLE patients showed glomerular deposition of MBL and MASP-1 without deposition of IgA2. No patient with other glomerulonephritides showed glomerular deposition of IgA1, IgA2, MBL and MASP-1. There was no correlation in clinical and pathological indicators between IgA2-positive and IgA2-negative patients with IgA GN. In conclusion, alternative pathway-mediated complement activation is associated in patients with mesangial deposition of IgA1 alone in IgA GN. In those with the deposition of both IgA1 and IgA2, both alternative and lectin pathways are activated, and mesangial deposition of IgA2 is associated with the lectin pathway-mediated complement activation in IgA GN.     

Renal tubular epithelial antigen-containing immune complexes stimulate interleukin-1 production by monocytes from patients with glomerulonephritis

We have investigated the effect of immune complexes (IC) derived from human renal tubular epithelial (RTE) antigen on the release of interleukin-1 (IL-1) in monocyte cultures from patients with glomerulonephritis (GN). When peripheral blood monocytes (PBM) were activated by IC, substantial amounts of IL-1 could be detected in the supernatants as measured by mouse thymocyte assay. The IC-induced IL-1 activity was significantly higher in patients with GN than in normal controls. To avoid the effect of prostaglandins on the IL-1 assay, we cultured PBM with addition of indomethacin and assayed IL-1 activity in the culture supernatants. This cyclooxygenase inhibitor augmented IC-induced IL-1 production. The results suggest that IC are involved in stimulating IL-1 production by PBM and thus play a role in the immune response in GN.     

Pretransplant Donor‐Specific HLA Class‐I and ‐II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q‐fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG‐DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q‐fixing DSA. Patients with both class‐I plus ‐II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q‐fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan‐IgG SAB assay are generally related to C1q‐fixation. We conclude that the presence of class‐I plus ‐II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q‐fixing IgG‐DSA could not be assessed due to their low prevalence.     

The role of hepatitis B surface antigen in the pathogenesis of glomerulopathies.

The frequency of hepatitis B surface antigen (HBsAg) has been studied in the sera and renal biopsies of 276 patients with various forms of glomerulonephritis (GN), the nephrotic syndrome and other nephropathies. Using a modified Hepanosticon method, HBs antigenemia was detected in 32 of 196 patients (16.3%) with immune complex (IC) GN and the nephrotic syndrome. Indirect immunofluorescence revealed HBsAg in 33 renal biopsy tissue specimens (16.8%). HBsAg was found in the sera of four of the 80 remaining patients with other renal diseases (5%), and in the renal biopsy tissues of another four (5%). Antibody against HBsAg could only be demonstrated in the serum of one glomerulonephritic patient. The sera of 18,799 normal blood donors were used as controls; of these 186 (0.99%) had positive tests for HBsAg. It is concluded that, in some patients with GN and the nephrotic syndrome, HBsAg-containing IC may be implicated in the development and/or progression of the disease.     

Nephritic factor: Description of a new quantitative assay and findings in glomerulonephritis.

A sensitive quantitative test for nephritic factor (NF) in human serum is reported. The test is based on the capacity of NF to initiate fluid phase consumption of the third complement (C) component in the presence of magnesium ions (Mg++) and of factors of the alternative pathway of C activation. These factors as well as C3 and C5 were supplied by the incorporation of normal human serum (NHS) into the assay mixture. In order to prevent C3 (and C5) consumption via the Ca++- and Mg++-dependent classical pathway, the test was performed in the presence of the chelating agent Mg-ethylene bis (oxyethylene-nitrilo) tetraacetic acid (Mg EGTA) which interacts preferentially with Ca++. The Mg EGTA concentration was found to be critical, a final concentration of 5 mM in the assay mixture being required for optimal results. By its heat stability (54 degrees to 56 degrees C, 30 min), NF could be distinguished from other, heat-labile NF-like factors. The NF test was applied to five categories of patients with glomerulonephritis (GN). Heat-stable NF activity was found in seven of 17 sera in the membranoproliferative glomerulonephritis (MPGN) group. Two of the 12 acute poststreptococcal GN sera had NF-like activity which disappeared upon heating. Serum C3 and proactivator (PA) concentrations varied widely in all groups but a clear positive relationship was found between the presence of NF and low serum C3 concentrations in MPGN. Renal immunofluorescence in MPGN indicated a lesser amount of lg deposited in glomeruli from patients with NF when compared to the NF-negative patients. Both groups had heavy C3 deposits. The availability of a sensitive, quantitative assay for NF may help to provide further insight into the various pathogenic mechanisms in different forms of MPGN.