Urinary C3 levels associated with sepsis and acute kidney injury-A pilot study.

Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 µg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine.

Differential urinary proteomics analysis of myocardial infarction using iTRAQ quantification.

Myocardial infarction (MI) is a disease characterized by high morbidity and mortality rates. MI biomarkers are frequently used in clinical diagnosis; however, their specificity and sensitivity remain unsatisfactory. Urinary proteome is an easy, efficient and noninvasive source to examine biomarkers associated with various diseases. The present study, to the best of the authors' knowledge, is the first to examine the urinary proteome using the isobaric tags for relative and absolute quantitation (iTRAQ) technology to identify potential diagnostic biomarkers of MI. The urinary proteome was analyzed within 12 h following the first symptoms of early­onset MI and at day 7 following percutaneous coronary intervention via iTRAQ labeling and two­dimensional liquid chromatography­tandem mass spectrometry. Candidate biomarkers were validated by multiple reaction monitoring (MRM) analysis. A total of 233 urinary proteins were differentially expressed. Gene enrichment analysis identified that the urinary proteome in patients with MI was associated with atherosclerosis, abnormal coagulation and abnormal cell metabolism. In total, 12 differentially expressed urinary proteins were validated by MRM analysis, five of which were associated with MI for the first time in the present study. Binary logistic regression analysis suggested that the combination of five urinary proteins (antithrombin­III, complement C3, α­1­acid glycoprotein 1, serotransferrin and cathepsin Z) may be used to diagnose MI with 94% sensitivity and 93% specificity. In addition, the protein expression levels of three proteins were significantly restored to normal levels following surgical treatment. The verified candidate biomarkers may be used for the diagnosis of MI, and for monitoring the disease status and the effects of treatments for MI. The present results may facilitate future clinical applications of the urinary proteome to diagnose MI.

Donor Urinary C5a Levels Independently Correlate With Posttransplant Delayed Graft Function.

BACKGROUND: Accumulating evidence implicates the complement cascade as pathogenically contributing to ischemia-reperfusion injury and delayed graft function (DGF) in human kidney transplant recipients. Building on observations that kidney injury can initiate in the donor before nephrectomy, we tested the hypothesis that anaphylatoxins C3a and C5a in donor urine before transplantation associate with risk of posttransplant injury. METHODS: We evaluated the effects of C3a and C5a in donor urine on outcomes of 469 deceased donors and their corresponding 902 kidney recipients in a subset of a prospective cohort study. RESULTS: We found a threefold increase of urinary C5a concentrations in donors with stage 2 and 3 acute kidney injury (AKI) compared donors without AKI (P < 0.001). Donor C5a was higher for the recipients with DGF (defined as dialysis in the first week posttransplant) compared with non-DGF (P = 0.002). In adjusted analyses, C5a remained independently associated with recipient DGF for donors without AKI (relative risk, 1.31; 95% confidence interval, 1.13-1.54). For donors with AKI, however, urinary C5a was not associated with DGF. We observed a trend toward better 12-month allograft function for kidneys from donors with C5a concentrations in the lowest tertile (P = 0.09). Urinary C3a was not associated with donor AKI, recipient DGF, or 12-month allograft function. CONCLUSIONS: Urinary C5a correlates with the degree of donor AKI. In the absence of clinical donor AKI, donor urinary C5a concentrations associate with recipient DGF, providing a foundation for testing interventions aimed at preventing DGF within this high-risk patient subgroup.

[Changes in the complement system in membranoproliferative glomerulonephritis]. / Izmeneniia v sisteme komplementa pri membranoproliferativnom glomerulonefrite.

AIM: To compare the clinical manifestations membranoproliferative glomerulonephritis (MPGN) in its idiopathic variant, lupus nephritis (LN), and C3 glomerulopathy (C3-GP), by comparing them with changes in the complement system. SUBJECTS AND METHODS: The clinic of nephrology followed up 42 patients with different types of MPGN in 2013 to 2015. The study included 35 patients divided into 3 groups: 1) 8 patients with C3-GP, 2) 13 with idiopathic MPGN; 3) 14 with Class IV LN. The investigators studied the blood and urine levels of components and markers for activation of the classical and alternative pathways (C3 and C4, С3а, C5a, CFH, CFB, and CFD) of the terminal complement complex (TCC). RESULTS: The detection rate of C3-GP was 19%. The patients with C3-GP were noted to have the lowest blood concentration of S3 and the highest urinary level of С3а, C5a, TCC, CFH, CFB, and CFD. C3 nephritic factor was detected in 2 patients from the C3-GP (dense deposit disease) group. CONCLUSION: Alternative complement pathway dysregulation caused by genetic or autoimmune factors plays a leading role in the pathogenesis of C3-GP.

Identification of urinary peptide biomarkers associated with rheumatoid arthritis.

Early diagnosis and treatment of rheumatoid arthritis are associated with improved outcomes but current diagnostic tools such as rheumatoid factor or anti-citrullinated protein antibodies have shown limited sensitivity. In this pilot study we set out to establish a panel of urinary biomarkers associated with rheumatoid arthritis using capillary electrophoresis coupled to mass spectrometry. We compared the urinary proteome of 33 participants of the Scottish Early Rheumatoid Arthritis inception cohort study with 30 healthy controls and identified 292 potential rheumatoid arthritis-specific peptides. Amongst them, 39 were used to create a classifier model using support vector machine algorithms. Specific peptidic fragments were differentially excreted between groups; fragments of protein S100-A9 and gelsolin were less abundant in rheumatoid arthritis while fragments of uromodulin, complement C3 and fibrinogen were all increasingly excreted. The model generated was subsequently tested in an independent test-set of 31 samples. The classifier demonstrated a sensitivity of 88% and a specificity of 93% in diagnosing the condition, with an area under the receiver operating characteristic curve of 0.93 (p<0.0001). These preliminary results suggest that urinary biomarkers could be useful in the early diagnosis of rheumatoid arthritis. Further studies are currently being undertaken in larger cohorts of patients with rheumatoid arthritis and other athridities to assess the potential of the urinary peptide based classifier in the early detection of rheumatoid arthritis.

C3 glomerulonephritis associated with monoclonal gammopathy: a case series.

BACKGROUND: C3 glomerulonephritis (GN) is a proliferative GN resulting from glomerular deposition of complement factors due to dysregulation of the alternative pathway of complement. Dysregulation of the alternative pathway of complement may occur as a result of mutations or functional inhibition of complement-regulating proteins. Functional inhibition of the complement-regulating proteins may result from a monoclonal gammopathy. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 32 Mayo Clinic patients with C3 GN, 10 (31%) of whom had evidence of a monoclonal immunoglobulin in serum. OUTCOMES: Clinical features, hematologic and bone marrow biopsy findings, kidney biopsy findings, kidney measures, complement pathway abnormalities, treatment, and follow-up of patients with C3 GN that was associated with a monoclonal gammopathy. RESULTS: Mean age of patients with C3 GN associated with monoclonal gammopathy was 54.5 years. Bone marrow biopsy done in 9 patients revealed monoclonal gammopathy of undetermined significance in 5 patients, small lymphocytic lymphoma/chronic lymphocytic leukemia in one patient, and no abnormal clones in the other 3 patients. Kidney biopsy showed membranoproliferative GN with bright capillary wall C3 staining in all 10 patients. Evaluation of the alternative pathway of complement showed abnormalities in 7 of 9 patients tested. No mutation in complement-regulating proteins was detected in any patient. As an index case, one patient with C3 GN and chronic lymphocytic leukemia was treated with rituximab, cyclophosphamide, vincristine, and prednisone, and one patient with C3 GN and monoclonal gammopathy of undetermined significance was treated with dexamethasone and bortezomib. Both patients showed significant decreases in hematuria and proteinuria and stabilization of kidney function. LIMITATIONS: Studies to show evidence of direct activation of the alternative pathway by monoclonal immunoglobulin were not done. CONCLUSIONS: The study highlights the association of C3 GN and monoclonal gammopathy, in particular in the older population, and the importance of targeting the underlying hematologic malignancy as an approach to treating C3 GN.

Crush syndrome after the Wenchuan earthquake: new experience with regional citrate anticoagulation continuous veno-venous hemofiltration.

PURPOSE: Acute renal failure (ARF) related to crush syndrome is usually treated with hemodialysis. Continuous veno-venous hemofiltration (CVVH) has seldom been adopted in this situation due to the main drawback of continuous anticoagulation. The purpose of this study was to evaluate the effectiveness and safety of regional citrate anticoagulation (RCA)-CVVH in two crush syndrome patients following the Wenchaun earthquake. METHODS: Two victims from the Wenchuan earthquake in Southwest China were admitted to our hospital on May 23, 2008, 11 days after their injury. The total entrapment time under the rubble was 5.5 and 22.5 hrs respectively. They remained oliguric on admission, in spite of vigorous treatment in the local hospital including aggressive fluid infusion, fasciotomy and intermittent hemodialysis. On admission, their serum myoglobin levels were 765 and 829 ng/mL, respectively. Further debridement and drainage were performed. RCA-CVVH was conducted; the citrate containing substitution fluid was infused in a pre-dilution manner at a rate of 4 l/h; calcium was infused through a separate access to the venous inlet of the double lumen catheter. The infusion rate was adjusted according to the serum ionized calcium and whole blood activated clotting time (WBACT). A low dose of low molecular weight heparin (LMWH) was infused at the rate of 150 approximately 300 U/h simultaneously for anticoagulation after anemia had been corrected and their wounds were stable. RCA-CVVH was substituted by conventional CVVH and LMWH anticoagulation when case 2 complicated with hypoxia. RESULTS: RCA-CVVH was well tolerated, hemodynamic status was stable, and no complications related with RCA-CVVH were noted. The body temperature and WBC decreased to normal range, while anemia and hypoalbuminia were corrected. The levels of serum myoglobin and creatine phosphokinase were also decreased to normal range. Their urine volume increased after 20 and 22 days of oliguria and the tubular function of the patients recovered well. Although the second case encountered acute cholecystitis and acute lung injury in the hospital, both the patients recuperated and neither of them underwent amputation. CONCLUSIONS: The present two crush patients have been successfully treated, but due to the limits of the small sample, it is difficult to generalize whether RCA-CVVH is safe enough for crush syndrome with a high risk of bleeding diathesis. Additional investigation with a larger number of patients is required. Fluid equilibrium, nutritional support, prevention of bleeding and infection are fundamental in this situation.

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli.

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).

Complement is activated in kidney by endotoxin but does not cause the ensuing acute renal failure.

BACKGROUND: Acute renal failure (ARF) in sepsis occurs when the release of multiple inflammatory mediators is induced by bacterial endotoxins. C3 mRNA is markedly up-regulated in mouse kidney after exposure to lipopolysaccharide (LPS). We hypothesized that LPS could induce tubular synthesis and secretion of C3, leading to activation of the complement cascade and direct renal tubular injury. METHODS: ARF was induced in mice by intravenous injection of LPS and was confirmed by an acute rise in blood urea nitrogen (BUN) and histologically by acute tubular necrosis. Three separate strategies were used to investigate the role of the complement system in this model of ARF: (1) Crry-Ig, a recombinant protein containing the potent murine complement C3 activation inhibitor Crry was injected at the same time as LPS (N = 8). (2) LPS was injected into transgenic mice overexpressing Crry in glomeruli and tubules (N = 8), and (3) LPS was injected into C3-deficient mice (N = 5). RESULTS: Compared with unmanipulated mice, C3 staining by immunofluorescence (IF) microscopy in mice injected with LPS was greater in renal cortical tubular cells (IF score of 2. 1 +/- 0.1 vs. 1.4 +/- 0.2 in controls, P = 0.013), most prominently at the basolateral surface. LPS injection led to a 16- to 42-fold increase in urinary C3 excretion. Despite reduction or complete elimination of renal C3 with maneuvers suppressing complement activation, BUN values were not statistically different across all groups. In no experiment did BUN values correlate with the extent of C3 staining. CONCLUSION: Although LPS up-regulates renal C3 synthesis, resulting in basolateral tubular C3 deposition, this is not responsible for LPS-induced ARF in mice.

[Protein markers in evaluation of nephroprotective effects of antihypertensive drugs in patients with arterial hypertension]. / Belkovye markery otsenki nefroprotektivnogo deistviia gipotenzivnykh sredstv u bol'nykh s arterial'noi gipertoniei.

Fifty patients with stable slight and moderate uncomplicated essential hypertension, treated by ramipril, atenolol, or isradipine, were examined. Total protein and urinary excretion of individual proteins were studied before and after treatment. Urinary concentrations of apolipoproteins A1 and B1, alpha 1-acid glycoprotein, alpha 1-antitrypsin, prealbumin, albumin, beta 2-microglobulin, transferrin, haptoglobin, IgG and IgA, and C3 and C4 complement components were measured. Index of proteinuria selectiveness was calculated for each portion of urine. All three drugs exerted a nephroprotective effect, atenolol being the most active of them. Apolipoproteins, IgG, and complement components were the most valuable for diagnosis. Their excretion correlated with the severity of arterial hypertension and efficiency of treatment. Use of protein markers helps reliably assess the renal function and monitor the treatment efficiency.

[Immune complexes and C3 and C4 of the complement in urine of patients with glomerulonephritis]. / Kompleksy immunologiczne oraz C3 I C4 dopelniacza w moczu chorych na klebuszkowe zapalenia nerek.

The circulating immune complexes (CIC), urinary immune complexes (UIC), C3 and C4 in urine (UC3 and UC4) in 99 patients with immunologic glomerular diseases: 13 with extracapillaris GN (ExGN), 38 with membranoproliferative GN (MPGN), 33 with mesangial proliferative GN (MesPGN), 5 with focal segmental glomerulosclerosis (FSGS), 5 with membranous nephropathy (MN), and 3 with minimal change nephropathy (MC) were investigated in the study. Depending on the (IS) immunosuppressive treatment all patients were classified into 3 groups. Group I: 61 recently biopsied patients with glomerular disease consisted of patients attending our renal department, group II: 24 patients with biopsy proven glomerular disease IS treated in the past but with GN with restrained disease activity, group III: 14 patients with active glomerulopathy who have been treated for some months. Nephelometry C1q binding test was used for CIC detection and 3.5% polyethylene glycol precipitate for its detection in urine by C1q binding test was applied. Radial immunodiffusion (NANORID) method was used for urine C3 and C4 detection. UC3 were detected in urine from 37% of all patients: in 39% patients of group I (GN at time of diagnosis), 38% of group II (GN with restrained disease activity) and 36% of group III (active GN received immunosuppressive therapy for several months). It suggest that nonimmunological-mechanism induce C3 detected in the rine of such patients. According to histological findings UC3 was detected in 3 patients with ExGN of group I, in patient with ExGN of group II and in about half patients with MPGN from group I and II, in about 25% patients with MesPGN from group I and II. About half patients with MesPGN of group III, one patient with MPGN of group III and a few patients with other histological findings of group I were UC3 positive. Simultaneous excretion of C4 in urine was detected in some UC3 positive patients (in about 5% patients). At the same time in about 50% UC3 positive patients was observed urinary excretion of IC. CIC and UIC were detected in 25% of all patients: in 29% of group I, in 17% of group II, and in 29% patients of group III. According to histological findings CIC was detected in 3 patients with ExGN, in 7 patients with MPGN from 26 of group I, and 3 from 7 with MPGN of group II, and 3 patients with MesPGN of group I and 1 patients with MesPGN of group II, and 33% patients with MesPGN of group III with high proteinuria. These findings suggested that urinary IC reflected the immunological activity of glomerulopathy and their presence in patients urine after IS treatment suggests incomplete response to this therapy while urinary C3 and C4 were connected with urinary protein excretion and may be of importance in tubulointerstitial injury and progression of renal insufficiency.

[Nephelometry as optic immunochemical method in the laboratory practice]. / Nefelometriia kak opticheskii immunokhimicheskii metod v laboratornoi praktike.

The authors offer a modification of the nephelometric method for clinical immunology laboratory to be used for measuring proteins (albumin, C3-complement, IgG, IgA, IgM, and alpha 2-macroglobulin) in human biological fluids. The original method making use of only Russian-made reagents and equipment has been used on a full scale for measuring the proteins in the blood serum, cerebrospinal fluid, urine, and saliva of normal subjects. The sensitivity and specificity of the method depend predominantly on the physicochemical parameters of the study.

Extensive complement activation in hereditary porcine membranoproliferative glomerulonephritis type II (porcine dense deposit disease).

Massive glomerular deposits of C3 and the terminal C5b-9 complement complex (TCC), but no immune complex deposits were detected by immunofluorescence in porcine membranoproliferative glomerulonephritis type II. TCC deposits were always observed with concomitant deposits of vitronectin (S-protein) in membranoproliferative glomerulonephritis, in contrast to a piglet with mesangial glomerulopathy where TCC was present without vitronectin co-deposition. Enzyme immunoassays revealed extensive systemic complement activation in 1-week-old affected piglets, observed by low plasma C3 (about 5% of normal) and high plasma TCC (about 10 x normal). Affected piglets revealed some plasma complement activation already at birth, 3 to 4 weeks before recognizable clinical disease. It is concluded that porcine membranoproliferative glomerulonephritis represents a nonimmune complex-mediated glomerulonephritis caused by unrestricted systemic complement activation with C3 consumption, TCC formation, and glomerular trapping of complement activation products. A pathogenetic mechanism of a defective or missing complement regulation protein is suggested.

Complement C3 fragments in urine: detection in systemic lupus erythematosus patients by western blotting.

We characterized urinary excretion of C3 fragments among patients with systemic lupus erythematosus (SLE) as a possible indicator of renal involvement. 28 patients, representing a broad range of disease activity were admitted to our study. Urinary proteins were separated on 4-20% gradient SDS-PAGE gels, under reducing conditions, and transblotted to nitrocellulose. Western blots were developed with a polyvalent goat-anti-human C3d antiserum, and an alkaline phosphatase-conjugated rabbit anti-goat IgG. Three patterns were obtained: 1) no bands detected; 2) bands suggesting the presence of intact C3; and 3) samples with additional low molecular (< 4 x 10(4)) bands. The 12 patients with no C3 bands had minimal disease activity (e.g. fatigue, arthralgia, arthritis, rash, oral ulcers). The seven patients with intact C3 patterns also had minimally active disease. Their primary clinical findings included fatigue, pleurisy, renal disease which had been treated, hemolytic anemia, and arthritis. Patients with low molecular weight C3 fragments in their urine formed two sub-sets, based upon their presenting features. The first group had severe disease and contained all patients with active lupus nephritis (n = 4), while the second consisted of non-renal patients with primary clinical findings of moderate disease activity (e.g. thrombocytopenia, pneumonitis, arthritis). Our results suggest urinary excretion of low molecular weight C3 fragments correlates with active renal disease, but is a variable finding among SLE patients with non-renal manifestations of disease activity.

[The effect of cyclosporin A on experimental glomerulonephritis in rabbits]. / Die Wirkung von Cyclosporin A auf die experimentelle Glomerulonephritis bei Kaninchen.

The effect of cyclosporin A on the development of the autologous phase of experimental rabbit glomerulonephritis was assessed. The glomerulonephritis was induced by an intravenous injection of duck anti-rabbit glomerular basement membrane globulin into adult rabbits. After 5-7 days, diffuse proliferative glomerulonephritis with proteinuria and linear glomerular basement membrane deposits of rabbit IgG were observed in all animals. The cyclosporin A treatment was started simultaneously with the injection of duck globulin. This attenuated the glomerular lesion, resulting in a normal urinary excretion. The serum level of anti-duck globulin antibody was reduced. In the group of rabbits, receiving an injection of duck anti-rabbit glomerular basement membrane globulin, cyclosporin A treatment and an additional high dose of rabbit anti-duck globulin 8 days later, equally no glomerulonephritis was observed. However, no effect of cyclosporin A treatment on the duck immunoglobulin induced passive Arthus phenomenon was seen.

Significance of urinary complement components in various glomerular diseases.

The concentrations of two components of the complement system (C1q and C3) were measured in the urine and blood in 10 normal subjects and 134 patients with primary and secondary glomerulonephritis by using a highly sensitive enzyme immunoassay. The values of urinary excretion of C1q and C3 were well correlated to the ratios of fractional clearance of these complement proteins to that of neutral dextran of 55 A, which was used to minimize the influences of glomerular sieving because of their comparable molecular size to these complement components. The rate of renal tubular reabsorption of C1q and C3 were at least 89.2 and 93.4% of filtrated C1q and C3, respectively. Urinary C1q and C3 were excreted significantly in cases of membranoproliferative glomerulonephritis (MPGN), membranous glomerulonephritis, IgA nephropathy with both mesangial and capillary immune complex (IC) deposit and also in case of active lupus nephritis. On the other hand, the concentrations of these complement components were low in case of minimal lesion nephrotic syndrome, mild proliferative glomerulonephritis, inactive lupus nephritis and diabetic nephropathy without any immune staining. There was a significant correlation between the urinary excretion of C1q or C3 and intraglomerular IC deposition, especially IC deposition along the glomerular capillary wall. However, the degree of the excretion of these proteins was not correlated to the degree or permselectivity of proteinuria. The correlations between urinary C1q and C3 were observed in cases of IgA nephropathy with both mesangial and capillary deposit and MPGN, although we couldn't see the correlation in the other glomerular diseases. It is suggested that urinary excretion of such complement components represents the fixation of complement by deposited intraglomerular IC. The measurement of urinary concentration of these complement components provides a new clue to investigation or diagnosis of glomerular diseases.

Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.