One-step detection of procollagen type III N-terminal peptide as a fibrosis biomarker using fluorescent immunosensor (quenchbody).

BACKGROUND: Procollagen type III N-terminal peptide (P-III-NP) is a fibrosis biomarker associated with liver and cardiac fibrosis. Despite the value of P-III-NP as a biomarker, its analysis currently relies on enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), which require more than 3 h. To facilitate early diagnosis and treatment through rapid biomarker testing, we developed a one-step immunoassay for P-III-NP using a quenchbody, which is a fluorescence-labeled immunosensor for immediate signal generation. RESULTS: To create quenchbodies, the total mRNA of P-III-NP antibodies was extracted from early-developed hybridoma cells, and genes of variable regions were obtained through cDNA synthesis, inverse PCR, and sequencing. A single-chain variable fragment (scFv) with an N-terminal Cys-tag was expressed in E. coli Shuffle T7, resulting in a final yield of 9.8 mg L-1. The fluorescent dye was labeled on the Cys-tag of the anti-P-III-NP scFv using maleimide-thiol click chemistry, and the spacer arm lengths between the maleimide-fluorescent dyes were compared. Consequently, a TAMRA-C6-labeled quenchbody exhibited antigen-dependent fluorescence signals and demonstrated its ability to detect P-III-NP at concentrations as low as 0.46 ng mL-1 for buffer samples, 1.0 ng mL-1 for 2 % human serum samples. SIGNIFICANCE: This one-step P-III-NP detection method provides both qualitative and quantitative outcomes within a concise 5-min timeframe. Furthermore, its application can be expanded using a 96-well platform and human serum, making it a high-throughput and sensitive method for testing fibrotic biomarkers.

Time to Change Our Viewpoints to Assess Renal Risks in Patients with Solitary Kidneys beyond Traditional Approaches?

Solitary functioning kidney (SFK) can be defined as the absence or hypofunction of a kidney due to acquired or congenital reasons. A congenital solitary functioning kidney (cSFK) is more common than is an acquired one (aSFK) and is characterized by the anatomical absence (agenesis) or hypofunction (hypoplasia; hypodysplasia) of one kidney from birth. Among the acquired causes, the most important is nephrectomy (Nx) (due to the donor, trauma or mass resection). Patients with SFK are at risk for the development of chronic kidney disease (CKD) in the long term. This risk potential is also significantly affected by hypertension. The relationship between hypertension and subclinical chronic inflammation is a connection that has not yet been fully clarified pathogenetically, but there are many studies highlighting this association. In recent years, studies examining different fibrosis and inflammation biomarkers in terms of the evaluation and prediction of renal risks have become increasingly popular in the literature. Oxidative stress is known to play an important role in homocysteine-induced endothelial dysfunction and has been associated with hypertension. In our study, we aimed to investigate the relationship between ambulatory blood pressure monitoring (ABPM) and urinary/serum fibrosis and inflammatory markers in patients with SFK. We prospectively investigated the relationship between ABPM results and soluble urokinase plasminogen activator receptor (suPAR), procollagen type III N-terminal peptide (PIIINP), homocysteine and other variables in 85 patients with SFK and compared them between cSFK and aSFK groups. In the etiology of SFK, a congenital or acquired origin may differ in terms of the significance of biomarkers. In particular, the serum homocysteine level may be associated with different clinical outcomes in patients with cSFK and aSFK.

Connection between Cardiac Fibrosis Biomarkers and Echocardiography Parameters in Advanced Chronic Kidney Disease Patients.

BACKGROUND: Myocardial fibrosis represents a mainstay pathway in the pathophysiology of uremic cardiomyopathy. This process leads to structural and functional changes in the heart, which can be detected by echocardiography. The purpose of our study was to determine the association between four echocardiographic parameters (ejection fraction (EF), global longitudinal strain (GLS), mean E/e' ratio, and left atrial volume indexed) and biomarkers associated with cardiac fibrosis, such as procollagen type I carboxy-terminal propeptide (PICP), procollagen type III N-terminal peptide (P3NP), and galectin-3 (Gal-3) in patients with end-stage renal disease (ESRD). METHODS: 140 patients with ESRD were enrolled and investigated by echocardiography and the serum levels of the aforementioned biomarkers were determined at baseline. RESULTS: The mean EF was 53.63 ± 8%, the mean GLS was -10.2 ± 5.3%, the mean E/e' ratio was 9.8 ± 4.3, and the mean left atrial volume indexed (LAVI) was 45.8 ± 14.2 mL/m2. The average levels for PICP, P3NP, and Gal-3 were 457.2 ± 240 µg/L, 242 ± 199.9 µg/L, and 10.7 ± 3.7 ng/mL, respectively. In regression analysis, PICP was strongly associated with all four echocardiographic parameters (EF: p = 0.0002, R2 = 0.69; GLS: p = 0.00001, R2 = 0.81; mean E/e': p = 0.00002; R2 = 0.89; LAVI: p = 0.003; R2 = 0.73). P3NP and Gal-3 were only associated with the EF (p = 0.01, R2 = 0.31 and p = 0.02; R2 = 0.35, respectively). CONCLUSION: Our study evidenced that PICP, a collagen-derived biomarker, is associated with important echocardiography parameters, suggesting that it can serve as an indicator of the presence of subclinical systolic and diastolic dysfunction in patients with advanced CKD.

Predictive Value of Collagen Biomarkers in Advanced Chronic Kidney Disease Patients.

Patients with chronic kidney disease have an increased risk of all-cause death. The value of collagen biomarkers such as procollagen type I carboxy-terminal propeptide (PICP) and procollagen type III N-terminal peptide (P3NP), in end-stage renal disease (ESRD), has not yet been defined (in the literature and in clinics). The purpose of this study was to determine the potential value of these new biomarkers in the prediction of mortality in this population. Plasma PICP and P3NP levels were determined in 140 patients with ESRD, not yet on dialysis, who were followed up for 36 ± 5.3 months. During follow-up, 58 deaths were recorded (41.4%), with the majority of them being cardiovascular deaths (43, 74.13%). Using the ROC curve, the cut-off value for the prediction of mortality for PICP was 297.31 µg/L, while for P3NP, the cut-off value was 126.67 µg/L. In univariate analysis, a value of PICP above the cut-off point was associated with a fivefold increased risk of mortality (hazard ratio (HR) 5.071, 95% confidence interval 1.935-13.29, p = 0.001) and a value of P3NP above the cut-off point was associated with a twofold increased risk of mortality (HR 2.089, 95% CI 1.044-4.178, p = 0.002). In a multivariable Cox proportional hazards model, PICP values remained independent predictors of mortality (HR 1.22, 95% CI 1.1-1.31, p < 0.0001). Our data suggest that the collagen biomarker PICP is an independent predictor of mortality in ESRD patients who are not yet on dialysis.

Association between plasma procollagen type III N-terminal peptide (P3NP) levels and physical performance in elderly men: The Korean Frailty and Aging Cohort Study (KFACS).

BACKGROUND: Physical performance decline associated with aging is clinically important in the development of disability in the older population. More recently, procollagen type III N-terminal peptide (P3NP) and synaptosomal-associated protein of 25 kDa (SNAP25) have been suggested as potential biomarkers for physical performance decline. OBJECTIVE: The objective of this pilot study was to examine plasma P3NP and SNAP25 levels in relation to muscle mass, strength, and performance status, and to investigate the association of plasma P3NP and SNAP25 levels with sarcopenia components. METHODS: Seventy-nine community-dwelling elderly men (mean age: 78.1 ± 3.5 years) were randomly selected and matched by age from the Korean Frailty and Aging Cohort Study. The sample was classified into the "normal," "low muscle mass only," "sarcopenia," and "low physical performance only" groups according to the criteria of the Asian Working Group for Sarcopenia 2019. Estimates and 95% confidence intervals (CIs) of log P3NP and log SNAP25 levels by muscle mass, strength, and performance status were obtained using a generalized linear model. Pearson correlations and multiple linear regression analyses were used to assess the association of log P3NP and log SNAP25 levels with appendicular skeletal muscle mass (ASM) index, handgrip strength, and physical performance. RESULTS: Log P3NP levels tended to be associated with low physical performance compared with the normal group (estimate = 0.54; 95% CI = -0.05, 1.14; p = 0.072). Log P3NP levels were inversely associated with physical performance (short physical performance battery and five-times sit-to-stand test) after adjusting for potential confounders (all p < 0.05) and tended to have an inverse association with gait speed (p = 0.078). Log P3NP levels tended to have a positive correlation with the ASM index (r2 = 0.042; p = 0.070), but not with handgrip strength (r2 = 0.0009; p = 0.795). However, no significant association between plasma SNAP25 levels and physical performance was observed. CONCLUSION: Plasma P3NP levels might be a potential biomarker for decreased physical performance in elderly men. Further studies with larger sample sizes are needed to confirm our findings.

Blood biomarker panel recommended for personalized prediction, prognosis, and prevention of complications associated with abdominal aortic aneurysm.

The aim of the study was to evaluate the ability of following biomarkers as diagnostic tools and risk predictors of AAA: C-reactive protein, interleukin-6, pentraxin-3, galectin-3, procollagen type III N-terminal peptide, C-terminal telopeptide of type I collagen, high-sensitive troponin I, and brain natriuretic peptide. Seventy-two patients with an AAA and 100 healthy individuals were enrolled into the study. We assessed individual biomarker performance and correlation between the AAA diameter and biomarker levels, and also, a multivariate logistic regression was used to design a possible predictive model of AAA growth and rupture risk. We identified following four parameters with the highest potential to find a useful place in AAA diagnostics: galectin-3, pentraxin-3, interleukin-6, and C-terminal telopeptide of type I. The best biomarkers in our evaluation (galectin-3 and pentraxin-3) were AAA diameter-independent. With the high AUC and AAA diameter correlation, the high-sensitive troponin I can be used as an independent prognostic biomarker of the upcoming heart complications in AAA patients. Authors recommend to add biomarkers as additional parameters to the current AAA patient management. Main addition value of biomarkers is in the assessment of the AAA with the smaller diameter. Elevated biomarkers can change the treatment decision, which would be done only based on AAA diameter size. The best way how to manage the AAA patients is to create a reliable predictive model of AAA growth and rupture risk. A created multiparameter model gives very promising results with the significantly higher efficiency compared with the use of the individual biomarkers.

The combination of 5 serum markers compared to FibroScan to predict significant liver fibrosis in patients with chronic hepatitis B virus.

BACKGROUND: We evaluated the performance of serum hyaluronan (HA), procollagen type III N-terminal peptide (PIIINP), type IV collagen (IVC), laminin (LN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), compared to transient elastography (FibroScan) in predicting significant liver fibrosis. METHODS: We therefore determined 4 serum fibrosis markers, FibroScan and liver biopsy in 70 consecutive adult patients with chronic hepatitis B. According to a modified Scheuer scoring system, significant fibrosis was defined as fibrosis stage ≥S2. We compared serum fibrosis markers to histological staging and FibroScan results using Spearman correlation analysis and area under receiver operating characteristic (ROC) curves (AUROCs). RESULTS: Of the 212 patients who had the results of FibroScans and four serum fibrosis markers for HBV, 70 had concurrent liver biopsy. Significant liver fibrosis was found in 24/70 patients. The serum levels of HA, PIIINP, IVC, LN, ALT, AST was all positively correlated with fibrosis stage of Liver biopsy. The coefficients with stages were respectively 0.468, 0.392, 0.538, 0.213, 0.350, 0.375. There was a significant difference between mild fibrosis (

Extracellular matrix turnover biomarkers predict long-term left ventricular remodeling after myocardial infarction: insights from the REVE-2 study.

BACKGROUND: Extracellular matrix turnover plays a key role in wound repair after myocardial infarction (MI). The aim of the study was to evaluate whether biomarkers of myocardial fibrosis measurements 1 month after MI may predict left ventricular (LV) remodeling. METHODS AND RESULTS: This prospective multicenter study included 246 patients with a first anterior Q-wave MI. Echocardiographic studies were performed at hospital discharge and 12 months after MI. Brain natriuretic peptide as well as biomarkers of myocardial fibrosis (type 1 collagen telopeptide, aminoterminal propeptide of type I procollagen, aminoterminal propeptide of type III procollagen) were measured 1 month after MI in 218 patients. In multivariate analysis, aminoterminal propeptide of type III procollagen/type 1 collagen telopeptide ratio ≤1 (odds ratio [95% confidence interval], 1.86 [1.02-3.39]; P=0.043) 1 month after MI and brain natriuretic peptide >100 pg/mL (2.35 [1.28-4.31]; P=0.006) were associated with a pejorative LV remodeling, whereas LV ejection fraction at discharge (per 5% increment; 0.78 [0.65-0.94]; P=0.01) was independently associated with lower rates of detrimental LV remodeling at 12 months. Patients with high brain natriuretic peptide and aminoterminal propeptide of type III procollagen/type 1 collagen telopeptide ratio ≤1, measured 1 month after MI, had the highest risk of developing a primary composite event (cardiovascular death or hospitalization for worsening heart failure; 14 events per 216 patients; P=0.0001) during a 3-year follow-up. CONCLUSIONS: Myocardial fibrosis turnover after MI is associated with LV remodeling. Low aminoterminal propeptide of type III procollagen/type 1 collagen telopeptide ratio (≤1) at 1 month is predictive, in addition to brain natriuretic peptide and LV ejection fraction, of detrimental LV remodeling as well as cardiovascular deaths and hospitalizations for heart failure.