Excretion of complement proteins and its activation marker C5b-9 in IgA nephropathy in relation to renal function.

BACKGROUND: Glomerular damage in IgA nephropathy (IgAN) is mediated by complement activation via the alternative and lectin pathways. Therefore, we focused on molecules stabilizing and regulating the alternative pathway C3 convertase in urine which might be associated with IgAN pathogenesis. METHODS: Membrane attack complex (MAC), properdin (P), factor H (fH) and Complement receptor type 1 (CR1) were quantified in urine samples from 71 patients with IgAN and 72 healthy controls. Glomerular deposition of C5, fH and P was assessed using an immunofluorescence technique and correlated with histological severity of IgAN and clinical parameters. Fibrotic changes and glomerular sclerosis were evaluated in renal biopsy specimens. RESULTS: Immunofluorescence studies revealed glomerular depositions of C5, fH and P in patients with IgAN. Urinary MAC, fH and P levels in IgAN patients were significantly higher than those in healthy controls (p < 0.001), but CR1 was significantly lower than that in healthy controls (p < 0.001). Urinary MAC and fH levels were positively correlated with serum creatinine (sCr), urinary N-acetyl-ß-D-glucosaminidase (u-NAG), urinary ß2 microglobulin (u-Bm), urinary protein (p < 0.001), interstitial fibrosis (MAC: p < 0.01, fH: p < 0.05) and the percentage of global glomerular sclerosis (p < 0.01). Urinary P was positively correlated with u-NAG, u-Bm, and urinary protein (p < 0.01). CONCLUSIONS: Complement activation occurs in the urinary space in IgAN and the measurement of levels of MAC and fH in the urine could be a useful indicator of renal injury in patients with IgAN.

The role of complement activation, detected by urinary C5b-9 and urinary factor H, in the excretion of urinary albumin in cisplatin nephropathy.

AIMS: Cis-diaminedichloroplatium II (CDDP) is an antineoplastic agent with serious renal toxicity, although the cause is not fully understood. The aim of this study was to clarify the functional roles of complement activation in cisplatin-nephropathy by examining the urinary complement components, C5b-9 and factor H. SUBJECTS: Five patients with advanced lung cancer were included in this study as they were due to receive CDDP or 1,1-cyclobutanedicarboxylatoplatinum II (CBDA). METHODS: Urine samples were collected before and after the chemotherapy for 13 days for measurements of C5b-9 (U-C5b-9), factor H (U-fH), albumin (U-Alb), beta2-microglobulin (U-beta2MG), and N-acetyl-beta-D-glucosamidase (NAG). RESULTS: The mean level of U-Alb during the 5 - 8 day period after CDDP treatment was significantly higher than before treatment (p < 0.01). There was no significant correlation between U-Alb and NAG (r = -0.031, p = 0.994), or U-Alb and U-beta2MG (r = 0.061, p = 0.978) during the 5 - 8 day after CDDP treatment. U-Alb, U-C5b-9 and U-fH clearly increased on Days 4 - 10 after CDDP treatment. In our three patients treated with CDDP, mean estimated glomerular filtration rate (eGFR) was slightly decreased at 7 and 13 days after the treatment, compared to that of pretreatment, whereas there was no difference of eGFR between 7 and 13 days. In patients treated with CBDA, these parameters were clearly at lower levels compared to those patients treated with CDDP. CONCLUSION: This study demonstrates that cisplatin may activate the complement pathway in the glomerulus, with factor H regulating the activation, resulting in decreased urinary albumin excretion and renoprotection.

[Evaluation of urinary factor H excretion in patients with idiopathic membranous nephropathy].

BACKGROUND: The complement system plays an important role in renal pathogenesis, and C5b-9, a terminal complement complex, is regarded as the principal mediator of proteinuria in idiopathic membranous nephropathy(MN). Since factor H regulates complement activation at the C3 step and is a crucial factor in complement-mediated tissue injury, the urinary excretion of factor H in patients with idiopathic MN was investigated. METHODS: Seven patients with biopsy-proven idiopathic MN were studied for twenty-four weeks. Urinary factor H levels were measured by ELISA from regularly collected urine samples, and then evaluated and compared with assays of urinary protein and C5b-9 excretion. RESULTS: During the study, five patients were treated with steroid therapy. All seven patients maintained stable renal function and showed a decline in urinary protein excretion. The mean level of urinary factor H was markedly elevated (156.1 +/- 47.1 U/mg U-Cr) before treatment (0 week), and gradually declined to 127.2 +/- 43.5 U/mg U-Cr at 12 weeks, and to 64.7 +/- 26.9 U/mg U-Cr) at 24 weeks. This followed decreases in urinary protein and urinary C5b-9 excretion. Percent change in urinary factor H level significantly decreased 24 weeks after treatment without affecting the plasma factor H level. CONCLUSION: These results suggest that factor H contributes to the regulatory mechanism of in situ complement activation, and thus the study of urinary factor H levels, as well as urinary C5b-9, may be significant in idiopathic MN.

Complement factor H as a marker for detection of bladder cancer.

BACKGROUND: The BTA TRAK and BTA stat tests for bladder cancer use monoclonal antibodies (mAbs) X13.2 and X52.1 to detect factor H (FH)-related material in urine. The exact ligands remain unknown. METHODS: Western blot analyses of purified FH, recombinant factor H-related protein 1 (FHR-1), and serum and urine samples were used to identify the ligands of X13.2 and X52.1. Recombinant FH constructs were used to identify the target sites of X13.2 and X52.1. To analyze whether natural ligands of FH could compete with its recognition by the capture mAb X52.1, we used surface plasmon resonance analysis. The role of the ligands of X52.1 in the BTA TRAK assay was tested with use of purified proteins and FH-depleted samples. RESULTS: X13.2 bound to domain 3 of FH and FH-like protein 1, whereas X52.1 bound to domain 18 of FH and to FHR-1. Using specific FH depletion from a bladder cancer patient's urine and purified FH, we demonstrated that FH is the ligand recognized by the BTA TRAK test. By contrast, FHR-1 in urine reduced the FH-dependent test signal. CONCLUSIONS: FH is a tumor marker for bladder cancer. To reveal the presence of bladder cancer, the BTA TRAK assay detects FH, whereas FHR-1 is able to partly inhibit this detection. This indicates a special mechanism for a diagnostic immunoassay based on the combined effect of simultaneous positive and negative signals in a single sample.

Glomerular deposition and urinary excretion of complement factor H in idiopathic membranous nephropathy.

BACKGROUND/AIMS: The complement system plays an important role in the pathogenesis of membranous nephropathy (MN). In order to elucidate the regulatory mechanism of complement activation, we demonstrated glomerular deposition and urinary excretion of complement factor H, which controls the alternative pathway and the amplification loop at the C3 step, in patients with idiopathic MN. METHODS: Renal biopsy specimens from 20 patients with idiopathic MN were studied immunohistochemically using monoclonal antibodies against complement components including factor H. SDS-PAGE and Western blotting analysis of urine samples were performed, and the urinary excretion of factor H and C5b-9 were measured by quantitative sandwich ELISA. RESULTS: Intense glomerular deposition of factor H was observed with C3b.C3c and C5b-9 at an early stage of the disease. Factor H was detected in Western blots of urine samples, but factor H-like protein 1 (FHL-1) was not. The mean level of urinary factor H was elevated (86.30 +/- 21.93 U/mg urinary creatinine) in comparison to that of normal controls (4.76 +/- 1.03 U/mg urinary creatinine). Urinary factor H level exhibited no correlation with clinical parameters; however, a negative correlation was found between urinary C5b-9/factor H and creatinine clearance (r = 0.662, p < 0.01). CONCLUSION: The source of glomerular and urinary factor H is supposedly a 150-kD protein. There was no evidence to suggest that FHL-1 is synthesized at the site of inflammation. The urinary C5b-9 to urinary factor H ratio is indicative of the degree of ongoing complement activation in the glomeruli and complement-mediated renal injury. These findings suggest that factor H contributes to the control mechanism of in situ complement activation and prevents renal damage in idiopathic MN.

Measurements of complement factor H-related protein (BTA-TRAK assay) and nuclear matrix protein (NMP22 assay)--useful diagnostic tools in the diagnosis of urinary bladder cancer?

Between 1997 and 2000 we investigated in a prospective study the voided urine samples of all consecutive patients undergoing cystoscopy independent from their clinical background (n = 705) with the BTA-TRAK assay (Bard Diagnostics, Redmont, USA) detecting a complement factor H-related protein (CFHrP) and the NMP22 assay (Matritech, Newton, USA) measuring a nuclear matrix protein, which is supposed to be specific for bladder cancer. The individuals were divided into three groups concerning the clinical background: 233 patients had urological diseases, 268 patients had urinary bladder cancer and 150 patients had other urological malignancies. Based on the clinical findings we compared our results with well established diagnostic methods for urinary bladder cancer such as cytology and the detection of hematuria. In addition, we investigated urine samples from 30 healthy individuals and 24 patients with urinary tract infection without performing cystoscopy. Following the recommendations of the European Group on Tumor Markers we used 95% specificity for benign urological diseases and urinary tract infections, which resulted in a sensitivity of 17% for active bladder cancer for the BTA-TRAK assay and 31% for NMP22. We compared these results with the detection of hematuria (specificity: 72%) and cytology, which had a sensitivity of 64% and 89%, respectively. Subsequently, we calculated sensitivity and specificity for the detection of relapse of the disease. Again using 95% specificity, in this case for patients with no evidence of disease (NED), in patients with recurrent disease the BTA-TRAK assay showed 8% sensitivity as compared to 12% for the NMP22 assay. Due to an insufficient specificity and sensitivity, both tests can neither be clinically useful in screening of high risk patients, nor in primary diagnosis of bladder cancer. They cannot replace neither cystoscopy nor cytology. In the follow-up care more investigations may be necessary to prove the benefit of existing diagnostic strategies for the discrimination between active and inactive bladder cancer.

Bladder tumor markers for monitoring recurrence and screening comparison of hyaluronic acid-hyaluronidase and BTA-Stat tests.

BACKGROUND: One of the goals of a noninvasive test for bladder carcinoma screening would be to reduce surveillance cystoscopies among patients with a history of bladder carcinoma. In addition, an accurate bladder carcinoma marker could be used to screen a high-risk population. The authors examined the efficacy of the hyaluronic acid-hyaluronidase (HA-HAase) and BTA-Stat tests to detect and predict bladder carcinoma recurrence and tested their specificity for bladder carcinoma screening. METHODS: Over a four year period, the authors prospectively collected 225 urine specimens from 70 bladder carcinoma patients and analyzed them by the HA-HAase test. Tumors were identified during 178 visits, and in 47 specimens there was no evidence of disease (NED). Twenty six of these 70 patients were randomly selected to have the BTA-Stat test (111 surveillance visits). In a separate study, 401 former Department of Energy (DOE) workers, who are likely to be at a higher risk for bladder carcinoma, were screened by the HA-HAase and BTA-Stat urine tests. RESULTS: The HA-HAase test had an approximately 91.0% sensitivity, 70% specificity, 87% accuracy, 92% positive predictive value (PPV), and 67% negative predictive value (NPV) in the 70 bladder carcinoma patients. There were 14 false-positives; however, 6 of these had recurred in approximately 5 months. Only 4 out of 33 NED cases recurred in that time period (chi-square = 5.43; degrees of freedom [DF] = 1; P = 0.0198). Thus, a false-positive HA-HAase test carried a significant risk of recurrence within five months (relative risk [RR] = 3.5; odds ratio [OR] = 5.44). In a direct comparison, the HA-HAase and BTA-Stat had 94% and 61% sensitivity, 63% and 74% specificity, 87% and 64% accuracy, 89% and 88% PPV, and 77% and 38% NPV, respectively. While 6 of the 10 false-positive on the HA-HAase test recurred in 5 months (chi-square = 9.6; DF = 1; P = 0.004), only 1 of the 7 false-positives on the BTA-Stat test recurred in that time period (chi-square = 0.096; DF = 1; P = 0.756). The RR and OR for the HA-HAase test were 10.2 and 24, and for the BTA-Stat, 1.4 and 1.5, respectively. In the DOE worker screening study, the HA-HAase and BTA-Stat had 14% (56 out of 401) and 16.7% (67 out of 401) positive rates, respectively. Sixty three percent of the positives on the BTA-Stat test, but only 25% of the positives on the HA-HAase test, had benign urologic conditions. None of the biomarker positive cases with clinical follow-up (n = 29) had evidence of bladder carcinoma. CONCLUSIONS: The HA-HAase test is efficient and superior to the BTA-Stat for detecting and predicting bladder carcinoma recurrence. Noninvasive tests with low false positive rates could be used for bladder carcinoma screening in high-risk populations (e.g., those with occupational exposure to carcinogens or smokers).

Qualitative and quantitative detection of urinary human complement factor H-related protein (BTA stat and BTA TRAK) and fragments of cytokeratins 8, 18 (UBC rapid and UBC IRMA) as markers for transitional cell carcinoma of the bladder.

OBJECTIVE: To evaluate the role of BTA stat, BTA TRAK, UBC Rapid, UBC IRMA and voided urinary cytology in the detection of bladder transitional cell carcinoma (TCC). METHODS: The study included 78 patients with TCC of the bladder (group A), 62 patients with a history of bladder TCC without tumor recurrence at the time of examination (B, control group), 20 patients with other malignancy of the urinary tract (C), 38 patients with non-malignant urinary tract diseases (D), 10 patients with urinary tract infection (E) and 10 healthy volunteers (F). Except in group F, voided urine was collected before cystoscopy or cystectomy. RESULTS: The specificity and sensitivity in bladder cancer detection were 87.1 and 74.4%, respectively with BTA stat, 79.3 and 48.7%, respectively with UBC Rapid, 100 and 33.3%, respectively with cytology, 72.6 and 75.6%, respectively with BTA TRAK, 64.5 and 70.5%, respectively with UBC IRMA. CONCLUSIONS: The BTA stat and BTATRAK tests are superior to UBC Rapid, UBC IRMA and urinary cytology in detection of bladder TCC. In daily practice however cytology remains the best adjunct to cystoscopy because of its high sensitivity in Tis and 100% specificity. Cystoscopy cannot be replaced by any of evaluated methods.

Effect of intravesical instillations on the human complement factor H related protein (BTA stat) test.

OBJECTIVES: The BTA stat is a rapid, non-invasive, qualitative urine test that detects bladder tumor-associated antigen (human complement factor H related protein) in urine. The sensitivity of this test is superior to that of urine cytology in detecting primary and recurrent tumors of the urinary bladder. Intravesical instillations are widely used to avoid recurrences and even progression. The objective of this study was to evaluate the effect of intravesical treatments on the BTA stat Test. METHODS: 501 consecutive patients followed up for bladder cancer were studied, of which 490 were eligible for analysis. Three hundred and twenty-seven (66.7%) of the patients had no history of intravesical treatments, whereas the remaining 163 (33.3%) had received treatments: 66 (40.5%) at the time of evaluation. A voided urine sample was obtained prior to cystoscopy and split for culture and BTA stat testing. The overall sensitivity and specificity were calculated and compared to the patients with no, past and present instillations. RESULTS: The overall sensitivity for the BTA stat Test was 56.6%, and the specificity was 76.4%. The specificity of the BTA stat Test was 80.7, 70.7 and 65.3% in those with no, past or present intravesical instillation treatments, respectively. The difference in specificity between those with no and present instillations was significant (p = 0.023), whereas the notable difference between those with no and past instillations did not reach significance (p = 0.076), nor was the difference between patients with past and present instillations significant (p = 0.558). Present instillation of mitomycin C had the strongest adverse effect on the test as the specificity was only 25.0%, whereas past treatment did not interfere with testing. The adverse effect of BCG treatment on testing extended. CONCLUSION: The overall specificity of the test is decreased in patients receiving intravesical treatments, whereas past treatments did not interfere with testing in general. However, the adverse effect of BCG on testing seems to extend, and therefore it is suggested that the BTA stat Test should not be used in patients having received BCG, and in those with present instillation of any type.

Human complement factor H related protein test for monitoring bladder cancer.

PURPOSE: The BTA stat test is a rapid, noninvasive, qualitative urine test that detects bladder tumor associated antigen (human complement factor H related protein) in urine. We compared BTA stat test to voided urine cytology in patients monitored for bladder cancer in a prospective trial, and determined whether this test is effective in detection of recurrence not seen by regular cystoscopy. MATERIALS AND METHODS: A total of 445 consecutive patients with bladder cancer were studied. A voided urine sample was obtained before cystoscopy and divided for culture, cytology and BTA stat testing. In cases of a positive BTA stat test but negative cystoscopy, excretory urography or renal ultrasound, random biopsies and collected ureteral urine samples for ureteral cytology were obtained. The overall sensitivity and specificity as well as positive and negative predictive values for BTA stat test, cytology and their combination were calculated. RESULTS: Of the 445 patients 118 (26.5%) had bladder cancer recurrence on cystoscopy, which was detected by BTA stat test and cytology in 63 (53.4%) and 21 (17.8%), respectively. Of the remaining 327 patients not having recurrent tumor on cystoscopy 81 (24.8%) had a positive BTA stat test. Excretory urography or renal ultrasound and random biopsies in 48 (59.3%) of these patients revealed 7 recurrences, making the total number of recurrent tumors 125 of 412 (30.3%). The overall sensitivities and specificities for the BTA stat test, cytology and their combination were 56.0%, 19.2%, 60.0% and 85.7%, 98.3% and 85.0%, respectively. CONCLUSIONS: The sensitivity for detection of recurrent tumor on BTA stat test is superior to that of voided urine cytology in all bladder cancer categories, whereas the specificity of voided urine cytology is higher than that for BTA stat test. However, a sixth of the patients with apparent false-positive BTA stat test results chosen for further investigation had recurrent tumors that were not found on routine cystoscopy. Although the sensitivity and specificity were highest when both tests were used, the differences were not significant overall. Therefore, the BTA stat test could potentially replace urine cytology for followup of superficial bladder cancer.

Qualitative determination of urinary human complement factor H-related protein (hcfHrp) in patients with bladder cancer, healthy controls, and patients with benign urologic disease.

OBJECTIVES: To assess the diagnostic quality of human complement factor H-related protein (hcfHrp, BTA STAT(TM)) as a qualitative urinary marker for bladder cancer. METHODS: Urine samples of 354 individuals (76 healthy volunteers, 111 patients with benign urologic disorders, 167 patients with histologically proven bladder cancer) were examined prior to therapy for the presence of hcfHrp. RESULTS: Overall test sensitivity was 62.9%. Sensitivity of low-grade/low-stage tumors was <50% and was thus comparable to published sensitivities of urinary cytology. In high-stage, high-grade tumors sensitivity was 100 and 77.3%, respectively. Superficial tumors with high risk of progression (pT1G3) were detected significantly better (88.9% sensitivity) than low-risk superficial tumors (pTa G1-3/pT1G1-2; 48.2%; p < 0.001). The overall specificity (healthy individuals and patients with benign urologic disease) was 93.0%. CONCLUSION: Since test sensitivity is comparable to urinary cytology and specificity is excellent, hcfHrp should be further evaluated in prospective studies focussing on the follow-up of patients with bladder cancer.

Comparison of two qualitative assays, the UBC rapid test and the BTA stat test, in the diagnosis of urothelial cell carcinoma of the bladder.

OBJECTIVES: To compare the diagnostic value of two rapid tests, the bladder tumor antigen (BTA stat) test and the newly developed urinary bladder cancer (UBC) Rapid test, in patients having symptoms suggestive of urothelial cell carcinoma (UCC) and patients being followed up after transurethral resection. METHODS: One hundred eighty patients with a mean age of 65.8 years (range 22 to 92) were included in the present study. The tests were performed on voided urine samples. Fifty-seven patients had symptoms suggestive of UCC and 123 patients were being followed up after complete transurethral resection of UCC. The voided urine was evaluated by the BTA stat and UBC Rapid test, which detects cytokeratins 8 and 18. All patients underwent subsequent cystoscopic evaluation and biopsy of any suspicious lesion. RESULTS: In 53 patients with histologically proved UCC, the BTA stat had a sensitivity of 52.8% and the UBC Rapid test of 66%. According to the histologic stage, the sensitivity of the BTA stat was 42.8% in pTa tumors, 61.5% in pT1, and 70% in pT2 or higher tumors. The sensitivity of the UBC test was 60.7% in pTa, 69. 2% in pT1, and 80% in pT2 or higher tumors. For histologic grades 1 to 3, the sensitivity was 38.8%, 52.6%, and 68.7% for the BTA stat and 44.4%, 78.9%, and 75% for the UBC Rapid test, respectively. The specificity was 70% and 90% for the BTA stat and UBC Rapid test, respectively. CONCLUSIONS: The UBC Rapid test was superior to the BTA stat in both sensitivity and specificity. Both assays are simple office procedures and require no special knowledge. However, they cannot replace, but only lower, the number of cystoscopies during the follow-up of patients with previous UCC of the bladder.

Multicenter trial of the quantitative BTA TRAK assay in the detection of bladder cancer.

BACKGROUND: Human complement factor H-related protein (hCFHrp) is produced by several bladder cancer cell lines and may be useful as a cancer marker. The aim of this study was to compare urinary hCFHrp and cytology for the detection of bladder cancer found by cystoscopy in patients with suggestive signs, symptoms, or preliminary test results. METHODS: The BTA TRAK assay, a quantitative enzyme immunoassay for the bladder tumor-associated antigen in urine, was compared with exfoliative cytology in 220 patients (155 men, 65 women; mean age, 64.2 years) presenting with signs, symptoms, or preliminary diagnostic results suggestive of this disease. Cystoscopy was the standard of detection. RESULTS: In the 100 patients found to have bladder cancer, the overall sensitivities of the BTA TRAK assay (at a previously determined decision threshold of 14 kilounits/L) and cytology were 66% (66 of 100) and 33% (33 of 100), respectively (P <0.001). The BTA TRAK assay proved to be statistically more sensitive than cytology for tumor grades I and II and for stage Ta and T1 tumors. In contrast, the overall specificity of the BTA TRAK assay in the 120 patients without cystoscopically confirmed bladder cancer was 69% (83 of 120) and that of cytology was 99% (119 of 120; P <0.001). The specificity of the BTA TRAK assay was higher in patients without benign or malignant genitourinary disease other than bladder cancer (76%; n = 89) than in patients with these conditions. When the BTA TRAK assay and cytology were used together such that a positive result in either test was scored as positive and the results compared with those of the BTA TRAK assay alone, increases in overall sensitivity and equivalent specificity were observed. CONCLUSION: Because of its relatively high sensitivity, the BTA TRAK assay could complement cytology as an adjunct to cystoscopy in the diagnosis and follow-up of most patients with bladder cancer.

Quantitative detection of human complement factor H-related protein in transitional cell carcinoma of the urinary bladder.

OBJECTIVE: The purpose of this study was to assess a new quantitative urinary tumor marker for transitional cell carcinoma of the urinary bladder (TCC), human complement factor H-related protein (hCFHrp, BTA TRAK). METHODS: Urine samples of 298 individuals (76 healthy volunteers, 118 patients with benign urologic disorders, 104 patients with histologically proven bladder cancer) were examined for the presence of hCFHrp. Samples of all patients were obtained prior to therapy. RESULTS: In comparison to healthy volunteers, patients with TCC had significantly higher urinary levels of hCFHrp (117.60 vs. 2.05 U/ml; p < 0.001). HCFHrp levels were positively correlated with tumor grade and stage. Patients with invasive TCC had significantly higher levels of hCFHrp than patients with superficial TCC (p = 0.001). Marker levels in superficial bladder cancer at high risk of tumor progression (pT1G3) were significantly higher as compared to low and intermediate grade superficial cancers. Elevated levels of hCFHrp were also found in patients with benign urologic disorders (median: 72.65 vs. 117.60 U/ml in cancer patients). Using a cutoff of 17.1 U/ml, hCFHrp had a sensitivity of 72.1% and, due to a high rate of false-positive determinations in patients with benign urologic disorders, a total specificity of 50.5%. CONCLUSIONS: HCFHrp (BTA TRAK) is a sensitive test for detection of bladder cancer and for identification of patients at high risk. Due to a high rate of false-positive results in patients with benign urologic diseases, the test should not be used in an unselected population.