Urinary collagen peptides: Source of markers for bone metabolic processes in kidney transplant recipients.

INTRODUCTION: Kidney transplant recipients (KTRs) are at an increased risk of fractures. Total urinary hydroxyproline excretion served as marker for bone resorption (BR) but was replaced by ß-CrossLaps (CTX), a C-terminal collagen α-1(I) chain (COL1A1) telopeptide. We investigated the low-molecular-weight urinary proteome for peptides associated with changes in bone metabolism after kidney transplantation. METHODS: Clinical and laboratory data including serum levels of CTX in 96 KTR from two nephrology centers were correlated with signal intensities of urinary peptides identified by capillary electrophoresis mass spectrometry. RESULTS: Eighty-two urinary peptides were significantly correlated with serum CTX levels. COL1A1 was the predominant peptide source. Oral bisphosphonates were administered for decreased bone density in an independent group of 11 KTR and their effect was evaluated on the aforementioned peptides. Study of the peptides cleavage sites revealed a signature of Cathepsin K and MMP9. Seventeen of these peptides were significantly associated with bisphosphonate treatment, all showing a marked reduction in their excretion levels compared to baseline. DISCUSSION: This study provides strong evidence for the presence of collagen peptides in the urine of KTR that are associated with BR and that are sensitive to bisphosphonate treatment. Their assessment might become a valuable tool to monitor bone status in KTR.

Increased Collagen Turnover Is a Feature of Fibromuscular Dysplasia and Associated With Hypertrophic Radial Remodeling: A Pilot, Urine Proteomic Study.

Fibromuscular dysplasia (FMD), a nonatherosclerotic, noninflammatory disease of medium-sized arteries, is an underdiagnosed disease. We investigated the urinary proteome and developed a classifier for discrimination of FMD from healthy controls and other diseases. We further hypothesized that urinary proteomics biomarkers may be associated with alterations in medium-sized, but not large artery geometry and mechanics. The study included 33 patients with mostly multifocal, renal FMD who underwent in depth arterial exploration using ultra-high frequency ultrasound. The cohort was separated in a training set of 23 patients with FMD from Belgium and an independent test set of 10 patients with FMD from Italy. For each set, controls matched 2:1 were selected from the Human Urinary Proteome Database. The specificity of the classifier was tested in 700 additional controls from general population studies, patients with chronic kidney disease (n=66) and coronary artery disease (n=31). Three hundred thirty-five urinary peptides, mostly related to collagen turnover, were identified in the training cohort and combined into a classifier. When applying in the test cohort, the area under the receiver operating characteristic curve was 1.00, 100% specificity at 100% sensitivity. The classifier maintained a high specificity in additional controls (98.3%), patients with chronic kidney (90.9%) and coronary artery (96.8%) diseases. Furthermore, in patients with FMD, the proteomic score was positively associated with radial wall thickness and wall cross-sectional area. In conclusion, a proteomic score has the potential to discriminate between patients with FMD and controls. If confirmed in a wider and more diverse cohort, these findings may pave the way for a noninvasive diagnostic test of FMD.

Selective Detection of Nano-Sized Diagnostic Markers Using Au-ZnO Nanorod-Based Surface-Enhanced Raman Spectroscopy (SERS) in Ureteral Obstruction Models.

BACKGROUND: This study investigated the diagnosis of renal diseases using a biochip capable of detecting nano-sized biomarkers. Raman measurements from a kidney injury model were taken, and the feasibility of early diagnosis was assessed. MATERIALS AND METHODS: Rat models with mild and severe unilateral ureteral obstructions were created, with the injury to the kidney varying according to the tightness of the stricture. After generating the animal ureteral obstruction models, urine was collected from the kidney and bladder. RESULTS AND DISCUSSION: After confirming the presence of renal injury, urine drops were placed onto a Raman chip whose surface had been enhanced with Au-ZnO nanorods, allowing nano-sized biomarkers that diffused into the nanogaps to be selectively amplified. The Raman signals varied according to the severity of the renal damage, and these differences were statistically confirmed. CONCLUSION: These results confirm that ureteral stricture causes kidney injury and that signals in the urine from the release of nano-biomarkers can be monitored using surface-enhanced Raman spectroscopy.

Blood and urinary collagen markers in osteoarthritis: markers of tissue turnover and disease activity.

Introduction: The need for diagnostic markers in osteoarthritis (OA) is acute and immediate, as sensitive and precise tools that monitor disease activity and treatment response are lacking. Collagens - types I, II, and III - are the skeleton of the extracellular matrix of joint tissues. Joint collagens are generally turned over at a low rate, but the balance between formation and degradation is disturbed, leading to the loss of, for example, cartilage.Areas covered: We discuss the markers reflecting collagen turnover and provide examples of how they have been applied in OA research, as well as how we believe these should be used in the future. We have searched PubMed for full-text articles written in English using different combinations of the following terms: OA, biomarker, and collagen. The result is a narrative review that gives examples from the literature.Expert opinion: Collagen markers show promise, as they are direct measures of tissue balance. Until now, collagen markers have mainly been tested in observational cohorts, which may provide insights into the association between the candidate marker and clinical variables; however, these do not advance the development of qualified markers that can be used for drug development or in clinical practice.

The Effect of a Low Glycemic Index Pulse-Based Diet on Insulin Sensitivity, Insulin Resistance, Bone Resorption and Cardiovascular Risk Factors during Bed Rest.

We determined the effects of a low glycemic-index pulse-based diet (i.e., containing lentils, chick peas, beans, and split peas) compared to a typical hospital diet on insulin sensitivity assessed by the Matsuda index from the insulin and glucose response to a two-hour oral glucose tolerance test, insulin resistance assessed by the homeostatic model assessment of insulin resistance (HOMA-IR), bone resorption assessed by 24 h excretion of urinary n-telopeptides(Ntx) and cardiovascular risk factors (blood lipids, blood pressure, arterial stiffness and heart rate variability) during bed rest. Using a randomized, counter-balanced cross-over design with one-month washout, six healthy individuals (30 ± 12 years) consumed the diets during four days of bed rest. The Matsuda index, HOMA-IR, urinary Ntx and cardiovascular risk factors were determined at baseline and after the last day of bed rest. Compared to the typical hospital diet, the pulse-based diet improved the Matsuda index (indicating increased insulin sensitivity; baseline to post-bed rest: 6.54 ± 1.94 to 6.39 ± 2.71 hospital diet vs. 7.14 ± 2.36 to 8.75 ± 3.13 pulse-based diet; p = 0.017), decreased HOMA-IR (1.38 ± 0.54 to 1.37 ± 0.50 hospital diet vs. 1.48 ± 0.54 to 0.88 ± 0.37 pulse-based diet; p = 0.022), and attenuated the increase in Ntx (+89 ± 75% hospital diet vs. +33 ± 20% pulse-based diet; p = 0.035). No differences for changes in cardiovascular risk factors were found between the two diet conditions, with the exception of decreased diastolic blood pressure during day three of bed rest in the pulse-based versus hospital diet (61 ± 9 vs. 66 ± 7 mmHg; p = 0.03). A pulse-based diet was superior to a hospital diet for maintaining insulin sensitivity, preventing insulin resistance, attenuating bone resorption and decreasing diastolic blood pressure during four days of bed rest.

Metabolomics reveals elevated urinary excretion of collagen degradation and epithelial cell turnover products in irritable bowel syndrome patients.

INTRODUCTION: Irritable bowel syndrome (IBS), the most commonly diagnosed functional gastrointestinal (GI) disorder in developed countries, is characterized by chronic abdominal pain, and altered bowel habits. OBJECTIVES: Accurate and timely diagnosis is challenging as it relies on symptoms and an evolving set of exclusion criteria to distinguish it from other related GI disorders reflecting a complex etiology that remains poorly understood. Herein, nontargeted metabolite profiling of repeat urine specimens collected from a cohort of IBS patients (n = 42) was compared to healthy controls (n = 20) to gain insights into the underlying pathophysiology. METHODS: An integrated data workflow for characterization of the urine metabolome with stringent quality control was developed to authenticate reliably measured (CV < 30%) and frequently detected (> 75%) metabolites using multisegment injection-capillary electrophoresis-mass spectrometry. Complementary statistical methods were then used to rank differentially excreted urinary metabolites after normalization to osmolality that were subsequently identified by high resolution tandem mass spectrometry and their electrophoretic migration behavior. RESULTS: Our work revealed ten consistently elevated urinary metabolites in repeat samples collected from IBS patients at two different time points (q < 0.05 after age and Benjamini-Hochberg/FDR adjustment), which were associated with greater collagen degradation and intestinal mucosal turn-over processes likely due to low-grade inflammation. IBS-specific metabolites identified in urine included a series of hydroxylysine metabolites (O-glycosylgalactosyl-hydroxylysine, O-galactosyl-hydroxylysine, lysine), mannopyranosy-L-tryptophan, imidazole propionate, glutamine, serine, ornithine, dimethylglycine and dimethylguanosine. A major limitation in this retrospective case-control study was significant co-morbidity of IBS patients with other illnesses, including depression and prescribed medications as compared to healthy controls. CONCLUSION: This work provides new mechanistic insights into the pathophysiology of IBS while also offering a convenient way to monitor patient disease progression and treatment responses to therapy based on a panel of urinary metabolites that avoids invasive blood sampling, colonoscopy and/or tissue biopsies.

Calcium intake in winter pregnancy attenuates impact of vitamin D inadequacy on urine NTX, a marker of bone resorption.

PURPOSE: Pregnancy is characterised by increased bone turnover, but high bone turnover with resorption exceeding formation may lead to negative maternal bone remodelling. Recent studies are conflicting regarding the effect of calcium on skeletal health in pregnancy. The aim of this study was to examine the seasonal effect of serum 25-hydroxyvitamin D (25OHD) and dietary calcium on a marker of bone resorption. METHODS: This was prospective study of 205 pregnant women [two cohorts; early pregnancy at 13 weeks (n = 96), and late pregnancy at 28 weeks (n = 109)]. Serum 25OHD and urine cross-linked N-telopeptides of type I collagen (uNTX) were measured at both time points. Intakes of vitamin D and calcium were recorded using 3-day food diaries at each trimester. RESULTS: Compared to summer pregnancies, winter pregnancies had significantly lower 25OHD and significantly higher uNTX. Higher calcium intakes were negatively correlated with uNTX in winter, but not summer. In late pregnancy, compared to those reporting calcium intakes ≥1000 mg/day, intakes of <1000 mg/day were associated with a greater increase in uNTX in winter pregnancies than in summer (41.8 vs. 0.9%). Increasing calcium intake in winter by 200 mg/day predicted a 13.3% reduction in late pregnancy uNTX. CONCLUSIONS: In late pregnancy, during winter months when 25OHD is inadequate, intakes of dietary calcium <1000 mg/day were associated with significantly increased bone resorption (uNTX). Additional dietary calcium is associated with reduced bone resorption in late pregnancy, with greater effect observed in winter. Further research regarding optimal dietary calcium and 25OHD in pregnancy is required, particularly for women gestating through winter.

Association of kidney fibrosis with urinary peptides: a path towards non-invasive liquid biopsies?

Chronic kidney disease (CKD) is a prevalent cause of morbidity and mortality worldwide. A hallmark of CKD progression is renal fibrosis characterized by excessive accumulation of extracellular matrix (ECM) proteins. In this study, we aimed to investigate the correlation of the urinary proteome classifier CKD273 and individual urinary peptides with the degree of fibrosis. In total, 42 kidney biopsies and urine samples were examined. The percentage of fibrosis per total tissue area was assessed in Masson trichrome stained kidney tissues. The urinary proteome was analysed by capillary electrophoresis coupled to mass spectrometry. CKD273 displayed a significant and positive correlation with the degree of fibrosis (Rho = 0.430, P = 0.0044), while the routinely used parameters (glomerular filtration rate, urine albumin-to-creatinine ratio and urine protein-to-creatinine ratio) did not (Rho = -0.222; -0.137; -0.070 and P = 0.16; 0.39; 0.66, respectively). We identified seven fibrosis-associated peptides displaying a significant and negative correlation with the degree of fibrosis. All peptides were collagen fragments, suggesting that these may be causally related to the observed accumulation of ECM in the kidneys. CKD273 and specific peptides are significantly associated with kidney fibrosis; such an association could not be detected by other biomarkers for CKD. These non-invasive fibrosis-related biomarkers can potentially be implemented in future trials.

Diagnostic and prognostic role of urinary collagens in primary human bladder cancer.

Collagen type 4 alpha 1 (COL4A1) and collagen type 13 alpha 1 (COL13A1) produced by urothelial cancer cells support the vital oncogenic property of tumor invasion. We investigated the diagnostic and prognostic capability of COL4A1 and COL13A1 in voided urine and compared the observed values with those of fragments of cytokeratin-19 (CYFRA21-1), nuclear matrix protein 22 (NMP-22), and voided urine cytology in bladder cancer (BCa). We collected voided urine samples from 154 patients newly diagnosed with BCa, before surgery and from 61 control subjects. Protein levels of COL4A1, COL13A1, CYFRA21-1, and NMP-22 in urine supernatants were measured using enzyme-linked immunosorbent assays. Diagnostic performance and optimal cut-off values were determined by receiver operating characteristic analysis. Urine levels of COL4A1, COL13A1, the combined values of COL4A1 and COL13A1 (COL4A1 + COL13A1), and CYFRA21-1 were significantly elevated in urine from patients with BCa compared to the controls. Among these biomarkers, the optimal cut-off value of COL4A1 + COL13A1 at 1.33 ng/mL resulted in 57.4%, 83.7%, 56.1%, 80.7%, and 91.7% sensitivity for low-grade tumors, high-grade tumors, Ta, T1, and muscle invasive disease, respectively. We evaluated the prognostic value of preoperative urine levels in 130 non-muscle invasive BCa samples after the initial transurethral surgery. A high urinary COL4A1 + COL13A1 was found to be an independent risk factor for intravesical recurrence. Although these data need to be externally validated, urinary COL4A1 and COL13A1 could be a potential diagnostic and prognostic biomarker for BCa. This easy-to-use urinary signature identifies a subgroup of patients with a high probability of recurrence and progression in non-muscle invasive and muscle invasive BCa.

Alterations in urinary collagen peptides in lupus nephritis subjects correlate with renal dysfunction and renal histopathology.

BACKGROUND: The excessive accumulation of extracellular matrix (ECM) in the renal tubulointerstitium is a key component of chronic renal damage in lupus nephritis (LN) and a critical determinant of the disease progression to renal failure. Detection of fibrosis requires renal biopsy and is therefore limited by high risks associated with an invasive procedure. This study explores whether a unique LN urinary peptidome can be identified and whether LN-specific alteration reflects the underlying fibrogenic process of altered ECM turnover. METHOD: Urinary peptides were analyzed for 36 LN and 35 nonrenal systemic lupus erythematosus (SLE) subjects and 58 healthy volunteers (HVs). RESULTS: In total, 70 collagen and 230 noncollagen peptides were significantly changed between LN and nonrenal SLE and between LN and HV and defined as 'LN peptides'; 14 proteases associated with observed LN collagen peptides were identified and activities in 9 proteases were significantly different between LN and nonrenal SLE; 28 collagen peptides were correlated with at least one parameter of clinical renal dysfunction or histolopathology. CONCLUSION: Urinary peptidomic alterations likely reflect pathogenic pathways involving ECM turnover in LN kidneys and potentially could be developed as biomarkers to monitor renal disease progression.

Diastolic Left Ventricular Function in Relation to Urinary and Serum Collagen Biomarkers in a General Population.

Current knowledge on the pathogenesis of diastolic heart failure predominantly rests on case-control studies involving symptomatic patients with preserved ejection fraction and relying on invasive diagnostic procedures including endomyocardial biopsy. Our objective was to gain insight in serum and urinary biomarkers reflecting collagen turnover and associated with asymptomatic diastolic LV dysfunction. We randomly recruited 782 Flemish (51.3% women; 50.5 years). We assessed diastolic LV function from the early and late diastolic peak velocities of the transmitral blood flow and of the mitral annulus. By sequencing urinary peptides, we identified 70 urinary collagen fragments. In serum, we measured carboxyterminal propeptide of procollagen type 1 (PICP) as marker of collagen I synthesis and tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1), an inhibitor of collagen-degrading enzymes. In multivariable-adjusted analyses with Bonferroni correction, we expressed effect sizes per 1-SD in urinary collagen I (uCI) or collagen III (uCIII) fragments. In relation to uCI fragments, e' decreased by 0.183 cm/s (95% confidence interval, 0.017 to 0.350; p = 0.025), whereas E/e' increased by 0.210 (0.067 to 0.353; p = 0.0012). E/e' decreased with uCIII by 0.168 (0.021 to 0.316; p = 0.018). Based on age-specific echocardiographic criteria, 182 participants (23.3%) had subclinical diastolic LV dysfunction. Partial least squares discriminant analysis contrasting normal vs. diastolic LV dysfunction confirmed the aforementioned associations with the uCI and uCIII fragments. PICP and TIMP-1 increased in relation to uCI (p<0.0001), whereas these serum markers decreased with uCIII (p≤0.0006). Diastolic LV dysfunction was associated with higher levels of TIMP-1 (653 vs. 696 ng/mL; p = 0.013). In a general population, the non-invasively assessed diastolic LV function correlated inversely with uCI and serum markers of collagen I deposition, but positively with uCIII. These observations generalise previous studies in patients to randomly recruited people, in whom diastolic LV function ranged from normal to subclinical impairment, but did not encompass overt diastolic heart failure.

Bone remodeling markers as lithogenic risk factors in patients with osteopenia-osteoporosis.

PURPOSE: To analyze the presence of phosphocalcic metabolism disorders in patients with osteopenia-osteoporosis without nephrolithiasis with respect to a control group. METHODS: A cross-sectional study was conducted in patients with osteopenia-osteoporosis without nephrolithiasis (n = 67) in lumbar spine or femur and in a control group (n = 61) with no lithiasis or bone disorders. Blood bone markers, phosphocalcic metabolism, fasting urine, 24-h urine lithogenic risk factors, and densitometry were recorded in both groups. SPSS 20.0 was used for statistical analysis. RESULTS: In comparison with the controls, significantly higher blood calcium (9.27 ± 0.36 vs. 9.57 ± 0.38, p = 0.0001), intact parathormone (45.6 ± 14.9 vs. 53.8 ± 18.9, p = 0.008), and alkaline phosphatase (61.9 ± 20.9 vs. 70.74 ± 18.9, p = 0.014) levels were found in patients with osteopenia-osteoporosis. In the 24-h urine test, citrate (1010.7 ± 647.8 vs. 617.6 ± 315.8, p = 0.0001) and oxalate (28.21 ± 17.65 vs. 22.11 ± 16.49, p = 0.045) levels were significantly lower in osteopenia-osteoporosis patients than in controls, with no significant difference in calcium (187.3 ± 106.9 vs. 207.06 ± 98.12, p = 0.27) or uric acid (540.7 ± 186.2 vs. 511.9 ± 167.06, p = 0.35) levels. Patients with osteopenia-osteoporosis had significantly higher levels of lithogenic risk factors associated with bone remodeling, including significantly increased ß-crosslaps and osteocalcin values and higher ß-crosslaps/osteocalcin ratios. CONCLUSION: Patients with osteopenia-osteoporosis without nephrolithiasis showed phosphocalcic metabolism disorders as well as lower urinary citrate and higher ß-crosslaps/osteocalcin and fasting calcium/creatinine ratios, which would increase the risk of nephrolithiasis. Hence, prospective studies are warranted to evaluate the long-term risks.

Preoperative Urodynamic Parameters (Valsalva Leak Point Pressure and Maximum Urethral Closure Pressure), Urinary Collagen and Plasma Vitamin D Levels as Predictors of Mid Urethral Sling Surgery Outcome.

PURPOSE: To determine the best predictor of the mid urethral sling outcome we calculated the AUC of ROC curves of preoperative parameters, including Valsalva leak point pressure, maximum urethral closure pressure, urinary NTx (N-telopeptide of crosslinked type I collagen) and plasma vitamin D values (D2, D3 and D2 plus D3). MATERIALS AND METHODS: This was an ancillary study of TOMUS (Trial of Mid-urethral Slings) and the ValUE (Value of Urodynamics Evaluation) trial in which subjects underwent mid urethral sling surgery for stress urinary incontinence. Valsalva leak point pressure and maximum urethral closure pressure were measured in 427 subjects, whereas NTx, vitamin D2, vitamin D3 and vitamin D2 plus D3 levels were obtained from 150, 116, 115 and 116 subjects respectively. Outcome success was defined using identical outcome (subjective and objective) variables for all subjects. ROC curves with corresponding AUC values were compared. RESULTS: TOMUS and ValUE subjects were significantly different in age, body mass index, UDI (Urogenital Distress Inventory) scores. TOMUS subjects had a lower surgical success rate compared to ValUE subjects (66.3% vs 76.0%, p = 0.03). The AUC values of Valsalva leak point pressure, maximum urethral closure pressure, NTx, and vitamins D2, D3 and D2 plus D3 were 0.542, 0.561, 0.702, 0.627, 0.645 and 0.640, respectively. The AUC of NTx was significantly higher than the AUCs of Valsalva leak point pressure and maximum urethral closure pressure (p = 0.02 and 0.03, respectively). CONCLUSIONS: Urinary NTx was the best predictor of the mid urethral sling outcome. This test is not only noninvasive, it is also modifiable. Finding ideal modifiable risk factors prior to mid urethral sling surgery should be subject to future investigations.

Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

An oral cathepsin K inhibitor ONO-5334 inhibits N-terminal and C-terminal collagen crosslinks in serum and urine at similar plasma concentrations in postmenopausal women.

Relationships between the plasma concentration of a cathepsin K inhibitor (ONO-5334) and inhibition of bone resorption markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in serum and urinary NTX/creatinine and CTX/creatinine were examined in 10 postmenopausal women. The subjects received slow-release tablets of 100mg ONO-5534 under fasted or fed conditions in a study with a crossover design. Inhibition of serum NTX and CTX levels and plasma concentrations of ONO-5334 were monitored at 0, 24, 48 and 168 h after dosing. Changes in urinary NTX/creatinine and CTX/creatinine levels in second morning urine were evaluated on 0, 1, 2 and 7 days after dosing. Data were analyzed using sigmoid maximal drug effect (Emax) models. The maximal inhibition, estimated Emax values, were -31.8% for serum NTX, -53.1% for serum CTX, -67.2% for urinary NTX/creatinine, and -95.2% for urinary CTX/creatinine. The estimated half maximal effective plasma concentrations (EC50) of ONO-5334 and confidence intervals were 1.79 (1.01 to 3.16) ng/mL for serum NTX, 2.07 (1.63 to 2.62) ng/mL for serum CTX, 1.85 (1.30 to 2.61) ng/mL for urinary NTX/creatinine, and 1.98 (0.94 to 3.76) ng/mL for urinary CTX/creatinine. EC50 values for the four crosslinks did not significantly differ, as indicated by the overlapping 95% confidence intervals. The highest signal-to-noise ratio was achieved with serum CTX, and was 2-fold higher than that on serum NTX. Inhibition for serum NTX and CTX, and urinary NTX/creatinine and CTX/creatinine by ONO-5334 were all correlated with correlation coefficients ranging from 0.55 to 0.80. In conclusion, data of ONO-5334 slow-releasing tablets in postmenopausal women were well fitted in Emax model. In all measured telopeptides, the maximal inhibition was obtained at urinary CTX/creatinine level, but serum CTX had the highest signal-to-noise ratio. Inhibition for all measured telopeptides by ONO-5334 were all correlated. The estimated half maximal effective plasma concentrations were not significantly different between all measured telopeptides.

The importance of calciuria as lithogenic factors in patients with osteopenia/osteoporosis.

PURPOSE: Recurrent kidney stones are associated with bone mineral density loss, altered bone remodeling markers, hypercalciuria and increased in fasting calcium/creatinine ratio. The objective was to determine biochemical alterations in urine in patients with osteopenia/osteoporosis without calcium kidney stones compared with patients with calcium kidney stones. METHODS: This is a cross-sectional study including 142 patients who were divided in two groups: Group 1 (patients with recurrent calcium kidney stones) and Group 2 (patients with osteopenia/osteoporosis in the lumbar spine or hip). Analyses of bone mineral density, calcium-phosphorous and bone metabolism and lithogenic risk factors in fasting urine samples and 24-h urine samples were performed. Statistical analysis was carried out with SPSS 17.0. A p ≤ 0.05 was considered statistically significant. RESULTS: Patients in Group 2 presented greater loss of bone mineral density and more elevated alkaline phosphatase, iPTH, phosphorous and ß-crosslaps levels, as compared to patients in Group 1. However, Group 1 presented greater urine calcium, oxalate and uric acid and a higher proportion of hypocitraturia, hypercalciuria and hyperoxaluria, as compared to Group 2. Multivariate analysis revealed that advanced age and ß-crosslaps levels are risk factors for bone mineral density loss, while low urinary calcium excretion was protective against bone demineralization. CONCLUSION: Patients with osteopenia/osteoporosis without lithiasis present some urinary biochemical alterations. This would explain the lack of lithogenic activity, although low calcium excretion in 24-h urine samples is a protective factor against the loss of bone mineral density.

A calcium-collagen chelate dietary supplement attenuates bone loss in postmenopausal women with osteopenia: a randomized controlled trial.

Menopause leads to an increased risk for osteoporosis in women. Although drug therapies exist, increasing numbers of people prefer alternative therapies such as dietary supplements, for example, calcium, vitamin D, and collagen hydrolysates for the prevention and treatment of osteoporosis. We have previously shown that a 3-month intervention using a calcium-collagen chelate (CC) dietary supplement was efficacious in improving bone mineral density (BMD) and blood biomarkers of bone turnover in osteopenic postmenopausal women. This study reports the long-term efficacy of CC in reducing bone loss in postmenopausal women with osteopenia. Thirty-nine women were randomly assigned to one of two groups: 5 g of CC containing 500 mg of elemental calcium and 200 IU vitamin D (1,25-dihydroxyvitamin D3) or control (500 mg of calcium and 200 IU vitamin D) daily for 12 months. Total body, lumbar, and hip BMD were evaluated at baseline, 6 and 12 months using dual-energy X-ray absorptiometry. Blood was collected at baseline, 6 and 12 months to assess levels of blood biomarkers of bone turnover. Intent-to-treat (ITT) analysis was performed using repeated measures analysis of variance pairwise comparisons and multivariate analysis to assess time and group interactions. The loss of whole body BMD in women taking CC was substantially lower than that of the control group at 12 months in those who completed the study and the ITT analysis, respectively (CC: -1.33% and -0.33% vs. control: -3.75% and -2.17%; P=.026, P=.035). The CC group had significantly reduced levels of sclerostin and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) (P<.05), and higher bone-specific alkaline phosphatase/TRAP5b ratio (P<.05) than control at 6 months. These results support the use of CC in reducing bone loss in osteopenic postmenopausal women.

Effects of dietary protein and glycaemic index on biomarkers of bone turnover in children.

For decades, it has been debated whether high protein intake compromises bone mineralisation, but no long-term randomised trial has investigated this in children. In the family-based, randomised controlled trial DiOGenes (Diet, Obesity and Genes), we examined the effects of dietary protein and glycaemic index (GI) on biomarkers of bone turnover and height in children aged 5-18 years. In two study centres, families with overweight parents were randomly assigned to one of five ad libitum-energy, low-fat (25-30% energy (E%)) diets for 6 months: low protein/low GI; low protein/high GI; high protein/low GI; high protein/high GI; control. They received dietary instructions and were provided all foods for free. Children, who were eligible and willing to participate, were included in the study. In the present analyses, we included children with data on plasma osteocalcin or urinary N-terminal telopeptide of collagen type I (U-NTx) from baseline and at least one later visit (month 1 or month 6) (n 191 in total, n 67 with data on osteocalcin and n 180 with data on U-NTx). The level of osteocalcin was lower (29.1 ng/ml) in the high-protein/high-GI dietary group than in the low-protein/high-GI dietary group after 6 months of intervention (95% CI 2.2, 56.1 ng/ml, P=0.034). The dietary intervention did not affect U-NTx (P=0.96) or height (P=0.80). Baseline levels of U-NTx and osteocalcin correlated with changes in height at month 6 across the dietary groups (P<0.001 and P=0.001, respectively). The present study does not show any effect of increased protein intake on height or bone resorption in children. However, the difference in the change in the level of osteocalcin between the high-protein/high-GI group and the low-protein/high-GI group warrants further investigation and should be confirmed in other studies.

Stage-specific quantitative changes in renal and urinary proteome during the progression and development of streptozotocin-induced diabetic nephropathy in rats.

Diabetic nephropathy (DN) is a microvascular complication associated with diabetes causing slow deterioration of kidneys leading to end-stage renal disease. Timely intervention and diagnosis are crucial in order to ameliorate and halt the progression of DN. Current diagnosis of DN consists of urine assays for detection of microalbuminuria, which have inadequate specificity and sensitivity. Hence, there arises a need to discover stage-specific biomarkers which can aid in the early detection of DN and also in identifying the mechanisms underlying pathogenesis of DN. Therefore the present study was undertaken to identify the differentially expressed proteins in the urine and to examine the pattern of proteomic changes occurring in the rat kidneys during the course of progression of streptozotocin-induced model of DN in rats. Two-dimensional gel electrophoresis coupled to MALDI-TOF mass spectrometry was employed to identify the differentially expressed proteins under diabetic conditions. Among the identified proteins Calgranulin A and Calgranulin B appeared in the urinary proteome at the fourth week of induction of diabetes while we recorded a time-dependent decrease in the expression of major urinary protein (alpha 2u globulin) in the urine as well as kidneys of diabetic rats. Parallel monitoring of targeted proteomic changes in the renal proteome revealed an increase in histone H2B phosphorylation at serine14 along with a gradual decrease in Bcl-2 and MMP-13 expression during the course of progression and development of streptozotocin-induced DN.