Urinary collagen degradation products as early markers of progressive renal fibrosis.

BACKGROUND: Renal fibrogenesis is associated with increased ECM remodeling and release of collagen fragments in urine in progressive renal disease. We investigated the diagnostic value of urinary collagen degradation products in a proteinuria-driven fibrosis rat model with and without anti-fibrotic S1P-receptor modulator FTY720 treatment. METHODS: Proteinuria was induced in male Wistar rats by Adriamycin (ADR) injection (n = 16). Healthy rats served as controls (n = 12). Six weeks post-injection, all underwent renal biopsy, and FTY720-treatment started in ADR-rats (n = 8) and controls (n = 6). Others remained untreated. Rats were sacrificed after 12 weeks. Collagen type I (C1M) and III (C3M) degradation fragments were measured in blood and urine using ELISA. Kidneys were stained for various inflammatory and fibrotic markers. RESULTS: Six weeks post-injection proteinuria increased (versus controls, P < 0.001) and although no accumulation of interstitial renal collagen type III (iColl3) was observed at this time, urinary C3M (uC3M) and C1M (uC1M) were significantly increased (both P < 0.001). At 12 weeks, uC3M (P < 0.001) and uC1M (P < 0.01) further increased in ADR-rats versus controls, just as fibronectin, PDGF-ß receptor, hyaluronan (all P < 0.01), iColl3, PAS, myofibroblasts, macrophages and T-cells (all P < 0.05). FTY720-treatment reduced accumulation of immune cells, α-SMA+ myofibroblasts and PAS-score, but not iColl3 and uC3M. Correlation analyses indicated that uC3M and uC1M reflected and predicted tubulointerstitial fibrogenesis. CONCLUSIONS: These data displayed urinary collagen breakdown products as sensitive early markers of interstitial fibrosis, preceding histological fibrotic changes, which might replace the invasive renal biopsy procedure to assess fibrosis. Anti-fibrotic FTY720 intervention reduced some fibrotic markers without affecting collagen type III metabolism.

Urine proteome analysis in heart failure with reduced ejection fraction complicated by chronic kidney disease: feasibility, and clinical and pathogenetic correlates.

AIMS: Urine proteome analysis (UPA) has already provided accurate discriminatory patterns of urinary peptides for renal disease, coronary artery disease, and asymptomatic LV diastolic dysfunction. UPA has now been used to characterize a discriminatory peptide biomarker pattern and establish a diagnostic classifier for heart failure patients with reduced ejection fraction (HFrEF) in the presence of chronic kidney disease (CKD). METHODS AND RESULTS: We analysed the urine proteome profiles obtained by capillary electrophoresis online coupled to micro-TOF (time of flight) mass spectrometry of 126 individuals, 59 HFrEF patients and 67 controls matched for age, sex, and renal function. In total, 107 significant discriminatory peptides were identified and used to establish a support vector machine-based classifier that was successfully applied to a test set of 25 HFrEF patients and 33 controls, achieving 84% sensitivity and 91% specificity. The majority of sequenced peptides were fragments of collagen type I and III. CONCLUSION: UPA was able to identify a set of HFrEF-specific urinary peptide biomarkers on a background of CKD that were successfully utilized to establish a syndrome's classifier.

Serum and urine markers of collagen degradation reflect renal fibrosis in experimental kidney diseases.

BACKGROUND: The extent of renal fibrosis in chronic kidney disease (CKD) is the best predictor for progression of most renal diseases. To date, no established biomarkers of renal fibrosis exist. METHODS: We measured circulating and urinary-specific matrix metalloproteinase (MMP)-generated collagen type I and III degradation fragments (C1M and C3M) and an N-terminal propeptide of collagen III (Pro-C3), as markers of collagen type III production, in three rat models of CKD and fibrosis: renal mass reduction (5/6 nephrectomy), progressive glomerulonephritis (chronic anti-Thy1.1 nephritis) and adenine crystal-induced nephropathy. Healthy rats served as controls. RESULTS: In all three models, the animals developed significant CKD and renal fibrosis. Compared with healthy rats, serum C1M and C3M significantly increased in rats with 5/6 nephrectomy and adenine nephropathy (2- to 3-fold), but not with chronic anti-Thy1.1 nephritis. Urinary C1M and C3M levels increased 9- to 100-fold in all three models compared with controls. Urinary degradation markers correlated closely with renal deposition of collagen type I and type III. Pro-C3 was significantly increased only in the urine of 5/6 nephrectomy rats. CONCLUSIONS: In particular, urinary markers of MMP-driven collagen degradation, rather than collagen production markers, may represent a novel, specific and non-invasive diagnostic approach to assess kidney fibrosis.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

OBJECTIVE: Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine. DESIGN AND METHODS: A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice. RESULTS: The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice. CONCLUSION: We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.

Measurement of matrix metalloproteinase 9-mediated collagen type III degradation fragment as a marker of skin fibrosis.

BACKGROUND: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies. METHODS: Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels. RESULTS: CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65). CONCLUSION: Increased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice.

Triple pharmacological blockade of the renin-angiotensin-aldosterone system in nondiabetic CKD: an open-label crossover randomized controlled trial.

BACKGROUND: Agents inhibiting the renin-angiotensin-aldosterone (RAAS) system have an important role in slowing the progression of chronic kidney disease. We evaluated the hypothesis that the addition of an aldosterone receptor antagonist to an angiotensin-converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT-1) receptor blocker (ARB) (triple RAAS blockade) may provide an additional benefit compared with an ACE inhibitor and ARB (double RAAS blockade). DESIGN: Randomized open controlled crossover study. SETTING & PARTICIPANTS: 18 whites (7 women, 11 men) from the Outpatient Department of Nephrology with chronic nondiabetic proteinuric kidney diseases, mean age 42.4 +/- 1.9 years (SEM). INTERVENTIONS: In the 8-week run-in period, all participants received the ACE inhibitor cilazapril (5 mg), the ARB telmisartan (80 mg), and the diuretic hydrochlorothiazide (12.5 mg) as double RAAS blockade to achieve the target blood pressure of less than 130/80 mm Hg. Participants were then randomly assigned to 2 treatment sequences, either the addition of spironolactone (25 mg) (triple RAAS blockade) through 8 weeks followed by double RAAS blockade through 8 weeks (sequence 1) or double RAAS blockade followed by triple RAAS blockade (sequence 2). MAIN OUTCOME MEASURES: 24-hour urine protein excretion (primary end point) and markers of tubular injury and fibrosis (secondary end points). Analysis was performed using analysis of variance for repeated measurements. RESULTS: At baseline, mean serum creatinine level was 1.16 +/- 0.09 mg/dL (103 +/- 8 micromol/L), estimated glomerular filtration rate was 107.8 mL/min (95% confidence interval, 93 to 140.9 [1.8 mL/s; 95% confidence interval, 1.55 to 2.35; Cockcroft-Gault formula), and 24-hour mean proteinuria was 0.97 +/- 0.18 g. Mean urine protein excretion was 0.7 g/24 h (95% confidence interval, 0.48 to 0.92) less after triple RAAS blockade than after double RAAS blockade (P = 0.01), without change in blood pressure. Urine excretion of N-acetyl-beta-d-glucosaminidase (P = 0.02) and amino-terminal propeptide of type III procollagen (P = 0.05) also significantly decreased. Potassium levels increased significantly after triple therapy (P = 0.02). However, no patient was withdrawn because of adverse effects. LIMITATIONS: Absence of blinding, small sample size, short treatment period, absence of histological assessment. CONCLUSIONS: Administration of an aldosterone receptor antagonist in addition to double RAAS blockade with an ACE inhibitor and ARB may slow the progression of chronic kidney disease. Additional studies are necessary to confirm this result.