Ability of a Urine Assay of Type II Collagen Cleavage by Collagenases to Detect Early Onset and Progression of Articular Cartilage Degeneration: Results from a Population-based Cohort Study.

OBJECTIVE: To evaluate the association of a sandwich assay for cartilage collagenase-mediated degradation, the C2C human urine sandwich assay (IB-C2C-HUSA), with early and late knee cartilage pathology and with progression of cartilage damage. METHODS: A population-based cohort with knee pain, age 40-79 years, was evaluated at baseline (n = 253) and after mean 3.3 years (n = 161). We evaluated the IB-C2C-HUSA and a related competitive inhibition assay (C2C). The C2C assay was applied to serum (sC2C) and urine (uC2C). Based on knee radiographs and magnetic resonance imaging (MRI), 3 subgroups [no cartilage pathology, preradiographic cartilage pathology, and radiographic osteoarthritis (ROA)] were evaluated cross-sectionally for association with biomarker levels. Longitudinally, we evaluated whether baseline assays predict subsequent progression of cartilage degeneration, defined by MRI cartilage loss. RESULTS: Cross-sectionally, statistically significant differences were seen in the 3 subgroups for IB-C2C-HUSA (p < 0.001), with the highest levels seen in ROA, and for sC2C (p = 0.023), while no differences were seen for uC2C (p = 0.501). Baseline IB-C2C-HUSA levels were higher in progressors vs nonprogressors (p = 0.003). In logistic regression analysis, only baseline IB-C2C-HUSA was associated with an increased risk of progression of cartilage damage (OR 1.78, 95% CI 1.03-3.09). CONCLUSION: The IB-C2C-HUSA degradation assay detects the generation of a pathology-related cartilage collagen peptide(s) that increase(s) with onset of degeneration of knee articular cartilage. The baseline values are associated with progression of cartilage degeneration over 3 subsequent years. This assay may have value in clinical OA trials. Further, it points to collagenase activity as a therapeutic target for controlling degeneration of articular cartilage.

[Biochemical indices of connective tissue metabolism in patients with chronic hepatitis B and C].

37 patients with chronic hepatitis B and C were examined. Patients were divided into 3 groups depending on the degree of connective tissue dysplasia. We investigated: free and protein-bounded hydroxyproline, collagenase activity, total alkaline phosphatase and its bone fraction, creatinine, calcium and phosphorus content in the blood serum and urine. It has been found the dependence of collagen synthesis from the state of connective tissue. The higher is the degree of dysplasia, the more intensive is the process of collagen synthesis (P < 0.05). The index of corellation between protein-bounded and free fraction can be used as a biochemical marker for determination the stage of pathological process in the liver and for monitoring the effectiveness of therapy.

Decreased collagen-degrading activity could be a marker of prolonged mesangial matrix expansion.

BACKGROUND: Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion. METHODS: We prepared two rat models, showing reversible or prolonged mesangial matrix expansion, induced by a single injection or two consecutive injections of anti-Thy-1.1 monoclonal antibody 1-22-3, respectively. We analyzed the glomerular expression of type I and type IV collagens; MMP-2, -9, and -13; membrane type 1-MMP (MT1-MMP); TIMP-1; and urinary type I collagen-degrading activity in both models. RESULTS: There were no differences in glomerular mRNA levels of type I and type IV collagens between the reversible and the prolonged models. MMP-9 mRNA expression and protein level was lower in the prolonged model than in the reversible one, whereas there were no differences in mRNA levels of MMP-2, -13, MT1-MMP, or TIMP-1 between the two models. Urinary type I collagen-degrading activity in the prolonged model was lower than that in the reversible one. Furthermore, there was a significant correlation between the mesangial matrix expansion and urinary type I collagen-degrading activity. CONCLUSIONS: Impaired expression of MMP-9 may contribute to the development of prolonged mesangial matrix expansion. Analysis of urinary type I collagen-degrading activity may provide additional diagnostic information in mesangial proliferative glomerulonephritis. Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion.

Matrix metalloproteinase 9 activity in urine of patients at risk for premature delivery.

A simple, noninvasive assay was used to quantitate urinary matrix metalloproteinase 9 activity among 15 patients with threatened premature labor. Both positive and negative predictive values for risk for premature delivery were 80%.

MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate.

Matrix metalloproteinases (MMPs) play an important role in many pathologic processes, but their activities are difficult to determine since no simple specific and/or chromogenic substrate exists. We have developed a novel MMP activity assay using a modified urokinase as a substrate. Protein engineering enabled the plasmin activation site in this urokinase to be substituted by a specific activation site recognized by MMPs. In this way the MMP activity can be monitored via urokinase activity as measured by a simple chromogenic assay. The assay was made specific for MMP-9 by a capture step using MMP-9-specific antibodies that do not interfere with MMP-activity. This assay monitors the amount of active enzyme as well as the latent, but potentially active proform. Using this assay the levels of MMP-9 were investigated in urine from patients with various kinds of carcinoma. High levels of both active and latent MMP-9 were detected in urine from patients with carcinoma of the bladder, whereas hardly any activity was observed in urine from healthy controls. MMP-9 in urine was present in its intact form. Surprisingly, MMP-9 was also increased in the urine of patients with nonurogenital carcinoma. Therefore, measurement of urinary MMP-9 activity levels may be a convenient diagnostic tool for various types of carcinoma.

Characterization of matrix metalloproteinases in human urine: alterations during adolescence.

Matrix metalloproteinases (MMPs) are a family of at least 14 zinc-dependent proteinases that have been implicated in matrix turnover under both normal and pathological conditions. Previous studies have shown that several MMPs are produced in various cell types in the kidney, suggesting that MMPs may be involved in renal morphogenesis and remodelling. Using a variety of techniques, including gelatin and casein zymography, gelatin affinity chromatography, immunoblotting, and immunoprecipitation, we have identified the major gelatinases in human urine as MMP-2 and MMP-9. Latent forms of both enzymes were detected in urine, as well as lower molecular mass species of each, consistent with activated forms of MMP-2 and MMP-9. MMP-2 and MMP-9 were also measured in individual human urine samples (n=40). No significant gender differences in MMP concentrations were detected. However, renal MMP expression appeared to be age dependent; the highest average amounts of urine MMP-2 were detected during adolescence, while the converse was true of urine MMP-9. Together, these findings indicate that under normal conditions, human urine contains MMP-2 and/or MMP-9, suggesting that these two MMPs are normally produced within the kidney, where they may regulate normal renal remodelling and matrix homeostasis in an age-specific manner.

Role of matrix metalloproteinase-9 in the basement membrane destruction of superficial urothelial carcinomas.

PURPOSE: This study was conducted to clarify which matrix metalloproteinases (MMPs) play a key role in destruction of the underlying basement membrane (BM) of superficial urothelial carcinomas. Urine concentrations of MMP-9 and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) were also measured. MATERIALS AND METHODS: Overexpression of MMP-1, MMP-2 and MMP-9 was analyzed immunohistochemically in 60 patients with transitional cell carcinomas of the urothelium (41 were pTa or pis, 19 were pT1-4), and compared them with type IV collagen expression in tumor BM. In 33 of them, urine concentrations of MMP-9 and TIMP-1 were measured by one-step sandwich enzyme immunoassay. RESULTS: Positive expression of MMP-1, MMP-2 and MMP-9 was found in 53%, 17%, and 65% of tumors, respectively. Only MMP-9 expression rates were increased with grades and stages (p = 0.03). In pTa and pis tumors, type IV collagen expression was reduced in 17 of 26 (65.4%), and it was associated with positive MMP-9 expression (p = 0.0283). MMP-9 was detected in all urine samples of urothelial cancer patients, while urine TIMP-1 was detectable in 18 of 33 patients. In 16 healthy volunteers, both of them were below detectable levels. Balance between urinary MMP-9 and TIMP-1 were particularly kept in superficial urothelial carcinomas with intact tumor BM. Tumor BM status, however, was not associated with urinary MMP-9 or TIMP-1 levels. CONCLUSIONS: These results suggest that MMP-9 plays a key role in the invasion step of superficial urothelial carcinomas. Detection of urinary MMP-9 may become a new, non-invasive mean for the diagnosis of urothelial carcinomas.

Matrix metalloproteinase-1 is induced by epidermal growth factor in human bladder tumour cell lines and is detectable in urine of patients with bladder tumours.

The matrix metalloproteinases are a family of enzymes that degrade the extracellular matrix and are considered to be important in tumour invasion and metastasis. The effect of epidermal growth factor (EGF) on matrix metalloproteinase-1 (MMP1) production in two human bladder tumour cell lines, RT112 and RT4, has been investigated. In the RT112 cell line, an increase in MMP1 mRNA levels was found after a 6-h incubation with EGF, and this further increased to 20-fold that of control levels at 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP2 mRNA levels remained constant over this time period, whereas in the RT4 cells no MMP2 transcripts were detectable, but MMP1 transcripts again increased with 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP1 protein concentration in the conditioned medium from both cell lines increased with 24- and 48-h treatment of the cells and the total MMP1 was higher in the medium than the cells, demonstrating that the bladder tumour cell lines synthesize and secrete MMP1 protein after continuous stimulation with EGF. MMP1 protein was detected in urine from patients with bladder tumours, with a significant increase in concentration with increased stage and grade of tumour. MMP1 urine concentrations may therefore be a useful prognostic indicator for bladder tumour progression.

Increased incidence of matrix metalloproteinases in urine of cancer patients.

Matrix metalloproteinases (MMPs) have been implicated in mechanisms of metastasis in experimental cancer models and in human malignancies. In this study, we used substrate gel electrophoresis (zymography) to determine the frequency of detection of MMPs in urine of patients with a variety of cancers. Three molecular weight classes of urinary MMPs, Mr 72,000, Mr 92,000, and high molecular weight (Mr > or = 150,000) species, were detected reproducibly and correlated with disease status. The Mr 72,000 and Mr 92,000 species were identified as MMP-2 and MMP-9, respectively, by Western blot analysis. The presence of biologically active MMP-2 (P < 0.001) or MMP-9 (P = 0.002) was an independent predictor of organ-confined cancer, and the high molecular weight species (P < 0.001) was an independent predictor of metastatic cancer. This is the first study to demonstrate that analysis of urinary MMPs may be useful in determining disease status in a variety of human cancers, both within and outside of the urinary tract.

Urine activity of cathepsin B, collagenase and urine excretion of TGF-beta 1 and fibronectin in membranous glomerulonephritis.

In 30% of cases nephrotic syndrome is caused by membranous glomerulonephritis (MG). Protein accumulation in glomeruli leads to progressive loss of kidney function and damage of structure in MG. The role of tissue proteolytic systems and growth factors in this process is not known. The purpose of the study was to estimate urine cathepsin B, collagenase activity and urine excretion of TGF-beta 1 and fibronectin in MG. Cathepsin B activity was greater in the urine of MG patients than in the control group (10.58 +/- 8.73 pmol AMC/mg creatinine per min-1 vs control 7.11 +/- 2.05 pmol AMC/mg creatinine per min-1; P < 0.05). Urine collagenase activity was higher in the group of patients than in the control group (8.59 +/- 4.26 pmol AMC/mg creatinine per min-1 vs control 3.84 +/- 2.09 pmol AMC/mg creatinine per min-1 P < 0.02). Urine excretion of fibronectin (45.60 ng/mg creatinine vs control 10.30 ng/mg creatinine; P < 0.04) and TGF-beta 1 levels in the urine were higher than in controls (283.55 +/- 248.13 pg/ml vs 36.11 +/- 48.01 pg/ml; P < 0.01). Results suggest glomerular overproduction of TGF-beta 1 and urinary leak of proteolytic enzymes (PE). This may result in decreased glomerular PE activity in MG and, with time, may lead to protein accumulation in renal glomeruli and to progressive loss of kidney function and damage of structures as the course of MG progresses. PE urine composition as well as ECM protein and cytokine urine excretion may allow noninvasive glomerulopathy course monitoring in humans in the future.

[Activity of cathepsin B and collagenase in urine and excretion of fibronectin and TGF-beta 1 in urine of patients with membranous glomerulonephritis]. / Aktywnosc katepsyny B i kolagenazy w moczu oraz wydalanie fibronektyny i TGF-beta 1 z moczem u chorych z bloniastym klebuszkowym zapaleniem nerek.

In 30% cases nephrotic syndrome is due to membranous glomerulonephritis (MG). Fifty percent of patients reveal end stage renal disease in 15 years follow-up. The another 50% gain persistent remission. The pathogenesis of disease is not known. Protein accumulation in glomeruli leads to progressive loss of kidney structure and function in MG. Also the role of tissue proteolytic systems and growth factors in this process is not known. We aimed to estimate urine cathepsin B, collagenase activity and urine excretion of TGF-beta 1 and fibronectin in MG. MG patients revealed increased urine cathepsin B activity (10.58 +/- 8.73 pmol AMC/mg creatinine/min. vs. control 7.11 +/- 2.05 pmol AMC/mg creatinine/min. [p < 0.05]), urine collagenase activity (8.59 +/- 4.26 pmol AMC/mg creatinine/min. vs. control 3.84 +/- 2.09 pmol AMC/mg creatinine/min. [p > 0.02]) and increased urine excretion of fibronectin (214 +/- 335 ng/mg creatinine vs. control 12.7 +/- 6.7 ng/mg creatinine [p < 0.05]) and increased urine excretion of TGF-beta 1 (283.55 +/- 248.13 pg/ml vs. control 36.11 +/- 48.01 pg/ml [p < 0.05]). The results indicates on glomerular overproduction of TGF-beta 1 and urinary leak of proteolytic enzymes which may exacerbate glomerular proteolytic activity in MG. This may lead to glomerular protein accumulation and progressive loss of kidney function and structure in MG. Increased urine fibronectin excretion in MG patients seems to confirm the hypothesis.

Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma.

Accumulating experimental evidence has linked the overproduction of extracellular matrix-degrading metalloproteinases with tumor cell invasion. In the present study one member of the metalloproteinase family, type IV collagenase (M(r) 72,000 gelatinase), is shown to be elevated in the urine of patients with transitional cell carcinoma of the bladder. The form of the enzyme in the urine was studied by three independent methods: enzyme-linked immunosorbent assay, Western immunoblotting; and gelatin zymography. Immunoblotting revealed that the enzyme was present as a series of fragments, each retaining the amino terminus of the mature proenzyme. A prominent M(r) 43,000 fragment was associated with the transitional cell carcinoma cases. Zymography demonstrated that multiple enzyme species with gelatinase activity were present in urine and that high-molecular-weight bands of substrate lysis corresponded to complexes between type IV collagenase and tissue inhibitor of metalloproteinases 2. The total amount of type IV collagenase antigen was significantly elevated in the urine of 37 transitional cell carcinoma patients (range, 0-1081 ng/ml; mean, 318.4 +/- 147.3) compared to 19 normal controls (P < or = 0.004) and 17 inflammatory disease controls (P < or = 0.011). Immunohistochemical staining of bladder tumor biopsies verified that the transitional cell carcinoma cells were producing the M(r) 72,000 enzyme. Thus, M(r) 72,000 type IV collagenase, which is present in the urine in many forms including fragments and complexes with inhibitors, may be a useful marker for bladder cancer diagnosis or prognosis.