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Background Diabetic retinopathy (DR) is a microvascular complication of diabetes highly associated to cardiovascular disease and diabetic kidney disease. However, these associations are not thoroughly investigated at an early type 2 diabetes disease stage. This study therefore evaluated the association between baseline DR status and development of cardiovascular events (CVEs), microalbuminuria, and kidney function decline and in persons with type 2 diabetes and normal urinary albumin excretion. Methods Post-hoc analysis of the PRIORITY study including 1758 persons with type 2 diabetes and normoalbuminuria followed for a median of 2.5 (IQR: 2.0–3.0) years. The study was originally designed to investigate a urinary proteomic risk classifier predictor of microalbuminuria development. DR at baseline was defined as non-proliferative and proliferative abnormalities, macular oedema, or history of laser treatment. Cox models were fitted to investigate the association of DR status with development of 1) a CVE composite defined as non-fatal myocardial infarction, stroke, coronary artery bypass graft, percutaneous coronary intervention, hospitalization for heart failure, or all-cause mortality; 2) persistent microalbuminuria (urinary albumin-creatinine ratio > 30mg/g); and 3) chronic kidney disease (CKD) G3 (eGFR 2). Models were adjusted for relevant risk factors. Results At baseline, 304 (17.3%) had DR. Compared to persons without DR, they were older (mean ± SD: 62.7 ± 7.7 vs 61.4 ± 8.3 years, p = 0.019), had longer diabetes duration (17.9 ± 8.4 vs. 10.6 ± 7.0 years, p 1c (62 ± 13 vs. 56 ± 12 mmol/mol, p

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. While a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal physiological consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild type kidney. Transmission electron microscopy (TEM) of Tshz3+/lacZ glomeruli revealed ultrastructural defects. Compared to wild type, Tshz3+/lacZ mice showed no difference in their urine parameters but lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild type (WT; control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Heterozygous mutations in HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease., Summary: A novel established mouse model carrying a heterozygous splicing human mutation in the Hnf1b gene exhibits phenotypes similar to those of patients with renal cysts and diabetes disease.

Background The hepatokine fetuin-A, released by the human liver, promotes pro-inflammatory effects of perivascular fat. The involvement of inflammation in type 2 diabetes mellitus (T2DM) can affect the kidney and contribute to the development of diabetic kidney disease. Therefore we examined the association of urinary fetuin-A protein fragments with renal damage in T2DM patients. Methods Urinary peptides of 1491 individuals using proteome data available from the human urine proteome database were analysed. Prediction of proteases involved in urinary peptide generation was performed using the Proteasix tool. Results We identified 14 different urinary protein fragments that belong to the region of the connecting peptide (amino acid 301–339) of the total fetuin-A protein. Calpains (CAPN1 and CAPN2), matrix metalloproteinase and pepsin A-3 were identified as potential proteases that were partially confirmed by previous in vitro studies. Combined fetuin-A peptides (mean of amplitudes) were significantly increased in T2DM patients with kidney disease and to a lesser extent with cardiovascular risk. Furthermore, fetuin-A peptide levels displayed a significant negative correlation with baseline estimated glomerular filtration rate (eGFR) values (r = −0.316, P Conclusions The urinary proteome analysis demonstrated the association of fetuin-A peptides with impaired kidney function in T2DM patients. Furthermore, fetuin-A peptides displayed early signs of kidney damage before albuminuria appeared and therefore can be used as markers for kidney disease detection.

Phenotype-specific omic expression patterns in people with frailty could provide invaluable insight into the underlying multi-systemic pathological processes and targets for intervention. Classical approaches to frailty have not considered the potential for different frailty phenotypes. We characterized associations between frailty (with/without disability) and sets of omic factors (genomic, proteomic, and metabolomic) plus markers measured in routine geriatric care. This study was a prevalent case control using stored biospecimens (urine, whole blood, cells, plasma, and serum) from 1522 individuals (identified as robust (R), pre-frail (P), or frail (F)] from the Toledo Study of Healthy Aging (R=178/P=184/F=109), 3 City Bordeaux (111/269/100), Aging Multidisciplinary Investigation (157/79/54) and InCHIANTI (106/98/77) cohorts. The analysis included over 35,000 omic and routine laboratory variables from robust and frail or pre-frail (with/without disability) individuals using a machine learning framework. We identified three protective biomarkers, vitamin D3 (OR: 0.81 [95% CI: 0.68-0.98]), lutein zeaxanthin (OR: 0.82 [95% CI: 0.70-0.97]), and miRNA125b-5p (OR: 0.73, [95% CI: 0.56-0.97]) and one risk biomarker, cardiac troponin T (OR: 1.25 [95% CI: 1.23-1.27]). Excluding individuals with a disability, one protective biomarker was identified, miR125b-5p (OR: 0.85, [95% CI: 0.81-0.88]). Three risks of frailty biomarkers were detected: pro-BNP (OR: 1.47 [95% CI: 1.27-1.7]), cardiac troponin T (OR: 1.29 [95% CI: 1.21-1.38]), and sRAGE (OR: 1.26 [95% CI: 1.01-1.57]). Three key frailty biomarkers demonstrated a statistical association with frailty (oxidative stress, vitamin D, and cardiovascular system) with relationship patterns differing depending on the presence or absence of a disability.

Although a rare disease, bilateral congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of end stage kidney disease in children. Ultrasound-based prenatal prediction of postnatal kidney survival in CAKUT pregnancies is far from accurate. To improve prediction, we conducted a prospective multicenter peptidome analysis of amniotic fluid spanning 140 evaluable fetuses with CAKUT. We identified a signature of 98 endogenous amniotic fluid peptides, mainly composed of fragments from extracellular matrix proteins and from the actin binding protein thymosin-β4. The peptide signature predicted postnatal kidney outcome with an area under the curve of 0.96 in the holdout validation set of patients with CAKUT with definite endpoint data. Additionally, this peptide signature was validated in a geographically independent sub-cohort of 12 patients (area under the curve 1.00) and displayed high specificity in non-CAKUT pregnancies (82 and 94% in 22 healthy fetuses and in 47 fetuses with congenital cytomegalovirus infection respectively). Change in amniotic fluid thymosin-β4 abundance was confirmed with ELISA. Knockout of thymosin-β4 in zebrafish altered proximal and distal tubule pronephros growth suggesting a possible role of thymosin β4 in fetal kidney development. Thus, recognition of the 98-peptide signature in amniotic fluid during diagnostic workup of prenatally detected fetuses with CAKUT can provide a long-sought evidence base for accurate management of the CAKUT disorder that is currently unavailable. ispartof: KIDNEY INTERNATIONAL vol:99 issue:3 pages:737-749 ispartof: location:United States status: published

Background and Aims The polymeric immunoglobulin receptor (pIgR) which transports immunoglobulins from the basolateral to the apical surface of epithelial cells was recently shown to be associated with kidney dysfunction. The immune defense is initiated at the apical surface where the N-terminal domain of pIgR, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to immunoglobulins. The aim of our study was to show the association of pIgR peptides with the cardio-renal syndrome in a large cohort and to get information on how the SC will be released. Method We investigated urinary peptides of 2707 individuals available in the Human Urine Proteome Database using capillary electrophoresis coupled to mass spectrometry. Results The mean abundance of 23 different pIgR peptides correlates negatively with the estimated glomerular filtration rate (eGFR, r=-0.314, p Conclusion In this large cohort, we could demonstrate that pIgR is associated with the cardio-renal syndrome and provide more detailed insights on how pIgR can be potentially cleaved to release the SC.

The polymeric immunoglobulin receptor (pIgR) transports immunoglobulins from the basolateral to the apical surface of epithelial cells. PIgR was recently shown to be associated with kidney dysfunction. The immune defense is initiated at the apical surface of epithelial cells where the N-terminal domain of pIgR, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to immunoglobulins. The aim of our study was to evaluate the association of pIgR peptides with the cardio-renal syndrome in a large cohort and to obtain information on how the SC is released. We investigated urinary peptides of 2964 individuals available in the Human Urine Proteome Database generated using capillary electrophoresis coupled to mass spectrometry. The mean amplitude of 23 different pIgR peptides correlated negatively with the estimated glomerular filtration rate (eGFR, rho = −0.309, p

Background: \ud Microalbuminuria is an early sign of kidney disease in people with diabetes and indicates increased risk of cardiovascular disease. We tested whether a urinary proteomic risk classifier (CKD273) score was associated with development of microalbuminuria and whether progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone.\ud \ud Methods: \ud In this multicentre, prospective, observational study with embedded randomised controlled trial (PRIORITY), we recruited people with type 2 diabetes, normal urinary albumin excretion, and preserved renal function from 15 specialist centres in ten European countries. All participants (observational cohort) were tested with the CKD273 classifier and classified as high risk (CKD273 classifier score >0·154) or low risk (≤0·154). Participants who were classified as high risk were entered into a randomised controlled trial and randomly assigned (1:1), by use of an interactive web-response system, to receive spironolactone 25 mg once daily or matched placebo (trial cohort). The primary endpoint was development of confirmed microalbuminuria in all individuals with available data (observational cohort). Secondary endpoints included reduction in incidence of microalbuminuria with spironolactone (trial cohort, intention-to-treat population) and association between CKD273 risk score and measures of impaired renal function based on estimated glomerular filtration rate (eGFR; observational cohort). Adverse events (particularly gynaecomastia and hyperkalaemia) and serious adverse events were recorded for the intention-to-treat population (trial cohort). This study is registered with the EU Clinical Trials Register (EudraCT 20120-004523-4) and ClinicalTrials.gov (NCT02040441) and is completed.\ud \ud Findings: \ud Between March 25, 2014, and Sept 30, 2018, we enrolled and followed-up 1775 participants (observational cohort), 1559 (88%) of 1775 participants had a low-risk urinary proteomic pattern and 216 (12%) had a high-risk pattern, of whom 209 were included in the trial cohort and assigned to spironolactone (n=102) or placebo (n=107). The overall median follow-up time was 2·51 years (IQR 2·0–3·0). Progression to microalbuminuria was seen in 61 (28%) of 216 high-risk participants and 139 (9%) of 1559 low-risk participants (hazard ratio [HR] 2·48, 95% CI 1·80–3·42; p

Chronic antibody-mediated rejection (cABMR) is the main cause of long-term renal graft loss. Late-stage diagnosis is made by detecting donor-specific antibodies (DSA) in blood combined with typical histomorphological lesions in renal allografts. There is a need for noninvasive biomarkers for cABMR that might permit screening and earlier diagnosis. In a case control study of 24 pediatric renal transplant recipients, urine samples were analyzed using capillary electrophoresis and mass spectrometry. Patients were matched with 36 pediatric renal transplant patients without cABMR. Statistical analysis used the nonparametric Wilcoxon test to identify 79 significant biomarkers, which were combined to a support vector machine-based classifier. After validation in an independent test cohort of eight pediatric patients with and 12 without cABMR, the area under the receiver operating characteristic (ROC) curve (AUC) for detection of cABMR was 0.92 (95% CI 0.71-0.99) with a sensitivity of 100% (95% CI 63-100%) and a specificity of 75% (95% CI 43-95%). Combining this classifier with the urinary proteomic marker CKD273 improved the detection of patients with cABMR with misclassification in only 2/20 of the patients. These data indicate that a biomarker pattern derived from urinary proteomics allows the detection of cABMR in pediatric renal transplant recipients with high sensitivity and moderate specificity.

Baroreflex activation therapy (BAT) is approved for the treatment of resistant hypertension. In addition to blood pressure (BP) reduction, pilot studies suggested several organoprotective effects of BAT. Thirty‐two patients with resistant hypertension were prospectively treated with BAT. Besides office BP and 24‐hour ambulatory BP (ABP) measurements, detection of a urinary proteome‐based classifier (CKD273), which has been shown to predict chronic kidney disease (CKD) progression, was carried out at baseline and after 6 months of BAT. Office BP significantly decreased from 170 ± 25/90 ± 18 to 149 ± 29/82 ± 18 mm Hg. Analysis of CKD273 score and eGFR with CKD‐EPI equation at baseline revealed strong correlation (r = 0.568, P

BACKGROUND: Chronic kidney disease (CKD) progression is currently assessed by a decline in estimated glomerular filtration rate (eGFR) and/or an increase in urinary albumin excretion (UAE). However, these markers are considered either to be late-stage markers or to have low sensitivity or specificity. In this study, we investigated the performance of the urinary proteome-based classifier CKD273, compared with UAE, in a number of different narrow ranges of CKD severity, with each range separated by an eGFR of 10 mL/min/1.73 m 2 . METHODS: A total of 2672 patients with different CKD stages were included in the study. Of these, 394 individuals displayed a decline in eGFR of >5 mL/min/1.73 m 2 /year (progressors) and the remaining individuals were considered non-progressors. For all samples, UAE values and CKD273 classification scores were obtained. To assess UAE values and CKD273 scores at different disease stages, the cohort was divided according to baseline eGFRs of ≥80, 70-79, 60-69, 50-59, 40-49, 30-39 and

ZusammenfassungChronische Nierenerkrankungen sind gekennzeichnet durch einen langsam fortschreitenden Verlust der Nierenfunktion, der schließlich zum Nierenversagen führt. Betroffene Patienten benötigen im Endstadium eine Nierenersatztherapie in Form der Dialyse und/oder Nierentransplantation mit erheblichen Konsequenzen sowohl für Mortalität als auch für die Lebensqualität der Patienten. Da die Prävalenz des Diabetes kontinuierlich deutlich steigt, nimmt gegenwärtig die diabetische Nephropathie (DN) als Dialyseursache zahlenmäßig den höchsten Rang ein. Die individuelle Behandlung der DN sowie der resultierenden kardiovaskulären Komplikationen in der frühen und damit einer Therapie zugänglichen Erkrankungsphase ist derzeit deutlich erschwert, da es an einer effektiven, nichtinvasiven, validen Routinediagnostik dieser Patienten mangelt. Die derzeit verwendeten Marker Serum-Kreatinin/eGFR und Albumin im Urin sind geeignet, um die DN in späteren Stadien abzubilden, jedoch sind sie von geringer Aussagekraft in der Erkennung von frühen Stadien. Eine neue diagnostische Methode ist die Analyse des Proteoms, der Gesamtheit aller Proteine und Peptide. Die Proteomanalyse hat die Identifikation von 273 Biomarkern im Urin zur Diagnostik chronischer Nierenerkrankungen ermöglicht. Ein auf diesen Biomarkern beruhender Klassifikator, CKD273, ermöglicht eine im Verhältnis zu den derzeit verwendeten Biomarkern signifikant bessere Identifizierung der DN. Die Daten zeigen vor allem eine Verbesserung der Früherkennung und Prognostik. Dies ermöglicht eine frühzeitige und gezielte Therapie und damit eine wesentlich verbesserte Option den fortschreitenden Verlust der Nierenfunktion aufzuhalten.

Pathophysiologische Mechanismen des Makulaodems infolge eines retinalen Venenverschlusses sind bis dato nicht vollstandig geklart. Die vorliegende klinisch-experimentelle Studie soll anhand laborchemischer Untersuchungen spezifische Proteine im Glaskorper von Patienten mit Venenastverschluss (VAV) identifizieren und ihre biologischen Funktionen analysieren. Unverdunnte Glaskorperproben wurden im Rahmen einer Kernvitrektomie von nicht vortherapierten Patienten mit VAV und Kontrollpatienten mit idiopathischen Glaskorpertrubungen entnommen und anhand Massenspektrometrie gekoppelt mit Kapillarelektrophorese (CE-MS) und gekoppelt mit Flussigkeitschromatographie (LC-MS/MS) untersucht. Signifikant dysregulierte Proteine wurden mit Enzyme-linked Immunosorbent Assay (ELISA) validiert. Grafische Darstellung der Protein-Protein-Interaktionen erfolgte anhand der String-Datenbank. 84 Proteine konnten bei 15 VAV-Patienten und 15 Kontrollen identifiziert werden. 14 dieser Proteine wiesen signifikant unterschiedliche Signalintensitaten zwischen beiden Gruppen auf. Sechs Proteine mit Signifikanzniveau

Introduction: Inter-individual variability in response to drug treatment has induced an increased demand for decisions via personalize medicine. Also, the contribution of proteomics to the era of personalized medicine would seem to be vital in improving therapeutic outcomes. Areas covered: We review validated biomarkers discovered by proteomics techniques and their use in personalized medicine with the focus on kidney diseases. We discuss this topic with a special emphasis on recent publications and relevant initiatives and depict some limitations that remain for personalized medicine. Expert opinion: The development of highly accurate biomarkers is essential for optimizing the management of kidney diseases. Various biomarkers of kidney diseases have been identified using proteomic techniques. However, only a few of these biomarkers showed the potential to be used in clinical practice concerning personalized medicine. Therefore, it becomes evident that the combination of multiple biomarkers confers higher accuracy and the ability to depict complex pathophysiological conditions, a prerequisite for personalized treatment. CKD273, a multimarker panel for early CKD detection may serve as a first example for personalized medicine in nephrology. Based on this successful example, proteomics is expected to develop into the key technology to guide personalized intervention.

International audience; Kidney function is altered by age together with a declined filtration capacity of 5-10% per decade after 35 years. Renal aging shares many characteristics with chronic kidney disease. Plasma levels of the bioactive peptide apelin also decline with age and apelin has been shown to be protective in chronic kidney disease. Therefore we evaluated whether apelin could also improve aging-induced renal lesions and function in mice. Since urine is for the major part composed of proteins and peptides originating from the kidney, we first studied apelin-induced changes, in the aging urinary peptidome. Despite the recently published age-associated plasma decrease of apelin, expression of the peptide and its receptor was increased in the kidneys of 24 months old mice. Twenty-eight days treatment with apelin significantly modified the urinary peptidome of 3 and 24 months old mice towards a signature suggesting more advanced age at 3 months, and a younger age at 24 months. The latter was accompanied by a decreased staining of collagen (Sirius red staining) in 24 months old apelin-treated mice, without changing aging-induced glomerular hypertrophy. In addition, apelin was without effect on aging-induced renal autophagy, apoptosis, inflammation and reduced renal function. In conclusion, treatment of aged mice with apelin had a limited effect on kidney lesions although modifying the urinary peptidome towards a younger signature. This supports evidence of apelin inducing more general beneficial effects on other aging organs, muscles in particular, as recently shown for sarcopenia, markers of which end up via the glomerular filtration in urine.

Renal Cysts and Diabetes Syndrome (RCAD) is an autosomal dominant disorder caused by mutations in the HNF1B gene encoding for the transcriptional factor hepatocyte nuclear factor-1B. RCAD is characterized as a multi-organ disease, with a broad spectrum of symptoms including kidney abnormalities (renal cysts, renal hypodysplasia, single kidney, horseshoe kidneys, hydronephrosis), early-onset diabetes mellitus, abnormal liver function, pancreatic hypoplasia and genital tract malformations. In the present study, using capillary electrophoresis coupled to mass spectrometry (CE-MS), we investigated the urinary proteome of a pediatric cohort of RCAD patients and different controls to identify peptide biomarkers and obtain further insights into the pathophysiology of this disorder. As a result, 146 peptides were found to be associated with RCAD in 22 pediatric patients when compared to 22 healthy age-matched controls. A classifier based on these peptides was generated and further tested on an independent cohort, clearly discriminating RCAD patients from different groups of controls. This study demonstrates that the urinary proteome of pediatric RCAD patients differs from autosomal dominant polycystic kidney disease (PKD1, PKD2), congenital nephrotic syndrome (NPHS1, NPHS2, NPHS4, NPHS9) as well as from chronic kidney disease conditions, suggesting differences between the pathophysiology behind these disorders.

Purpose Early and accurate detection of deep vein thrombosis (DVT) is an important clinical need. Based on the hypothesis that urinary peptides may hold information on DVT in conjunction with pulmonary embolism (PE), the study was aimed at identifying such peptide biomarkers using capillary electrophoresis coupled mass spectrometry. Experimental design Patients with symptoms of unprovoked/idiopathic DVT and/or PE were examined by doppler-sonography or angio-computed tomography. Urinary proteome analysis allowed for identification of respective peptide biomarkers. To confirm their biological relevance, we induced PE in mice and assessed human ex vivo thrombi. Results We identified 62 urinary peptides as DVT-specific biomarkers, i.e. fragments of collagen type I and a fragment of fibrinogen β-chain. The presence of fibrinogen α/β in the acute thrombus, and collagen type I and osteopontin in the older, organized thrombus was demonstrated. The classifier DVT62 established through support vector machine (SVM) modeling based on the 62 identified peptides was validated in an independent cohort of 47 subjects (six cases and 41 controls) with a sensitivity of 100% and specificity of 83%. Conclusions and clinical relevance Urine proteome analysis enabled the detection of DVT-specific peptides, which were validated in human and mouse tissue. Furthermore, it allowed for the establishment of an urinary-proteome based classifier that is relatively specific for DVT. The data provide the basis for assessment of these biomarkers in a prospective clinical study.

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Chronic kidney disease is associated with increased risk of CKD progression and death. Therapeutic approaches to limit progression are limited. Developing tools for the early identification of those individuals most likely to progress will allow enriching clinical trials in high risk early CKD patients. The CKD273 classifier is a panel of 273 urinary peptides that enables early detection of CKD and prognosis of progression. We have generated urine capillary electrophoresis-mass spectrometry-based peptidomics CKD273 subclassifiers specific for CKD stages to allow the early identification of patients at high risk of CKD progression. In the validation cohort, the CKD273 subclassifiers outperformed albuminuria and CKD273 classifier for predicting rapid loss of eGFR in individuals with baseline eGFR > 60 ml/min/1.73 m2. In individuals with eGFR > 60 ml/min/1.73 m2 and albuminuria 60 ml/min/1.73 m2. These CKD273 subclassifiers represented the earliest evidence of rapidly progressive CKD in non-albuminuric individuals with preserved renal function.

OncoTarget 6(33), 34106-34117 (2015). doi:10.18632/oncotarget.5896, Published by Impact Journals LLC, [S.l.]

In a previous cross-sectional study, we identified a multidimensional urinary classifier (HF1), which was associated with left ventricular dysfunction. We investigated whether HF1 predicts cardiovascular end points over and beyond traditional risk factors. In 791 randomly recruited Flemish (mean age, 51.2 years; 50.6% women), we quantified HF1 by capillary electrophoresis coupled with mass spectrometry. In addition, we measured cardiovascular risk factors. HF1 averaged −0.97 U (range, −3.26 to 2.60). Over 6.1 years (median), 35 participants died and 63, 45, and 22 experienced fatal or nonfatal cardiovascular, cardiac, or coronary events, respectively. The incidence of fatal combined with nonfatal cardiovascular and cardiac end points, standardized for sex and age, increased across thirds of the HF1 distribution ( P ≤0.014), whereas trends for all-cause mortality and coronary events were nonsignificant ( P ≥0.10). The multivariable-adjusted hazard ratios (+1-SD) were 1.30 (95% confidence interval, 1.03–1.65; P =0.029) and 1.39 (1.06–1.84; P =0.018) for cardiovascular and cardiac events in relation to HF1. For systolic pressure, the corresponding estimates were 0.97 (0.74–1.28; P =0.85) and 0.93 (0.67–1.29; P =0.66), respectively. The HF1 upper thresholds optimized by maximizing Younden’s index were −0.50 and −0.36 U for cardiovascular and cardiac end points, respectively. Prognostic accuracy significantly ( P ≤0.006) improved by adding HF1 to Cox models already including the other baseline predictors. Sensitivity analyses, from which we excluded 71 participants with previous cardiovascular disease, were confirmatory. In conclusion, over a 6-year period, the urinary proteome, but not systolic pressure, predicted cardiovascular and cardiac disease.

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease-associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE-MS), has been increasingly used for the detection of peptide biomarkers (

Background: Early prevention of diabetic nephropathy is not successful as early interventions have shown conflicting results, partly because of a lack of early and precise indicators of disease development. Urinary proteomics has shown promise in this regard and could identify those at high risk who might benefit from treatment. In this study we investigate its utility in a large type 2 diabetic cohort with normoalbuminuria.Methods: We performed a post hoc analysis in the Diabetic Retinopathy Candesartan Trials (DIRECT-Protect 2 study), a multi centric randomized clinical controlled trial. Patients were allocated to candesartan or placebo, with the aim of slowing the progression of retinopathy. The secondary endpoint was development of persistent microalbuminuria (three of four samples). We used a previously defined chronic kidney disease risk score based on proteomic measurement of 273 urinary peptides (CKD273-classifier). A Cox regression model for the progression of albuminuria was developed and evaluated with integrated discrimination improvement (IDI), continuous net reclassification index (cNRI) and receiver operating characteristic curve statistics.Results: Seven hundred and thirty-seven patients were analysed and 89 developed persistent microalbuminuria (12%) with a mean follow-up of 4.1 years. At baseline the CKD273-classifier predicted development of microalbuminuria during follow-up, independent of treatment (candesartan/placebo), age, gender, systolic blood pressure, urine albumin excretion rate, estimated glomerular filtration rate, HbA1c and diabetes duration, with hazard ratio 2.5 [95% confidence interval (CI) 1.4-4.3; P = 0.002] and area under the curve 0.79 (95% CI 0.75-0.84; P < 0.0001). The CKD273-classifier improved the risk prediction (relative IDI 14%, P = 0.002; cNRI 0.10, P = 0.043).Conclusions: In this cohort of patients with type 2 diabetes and normoalbuminuria from a large intervention study, the CKD273-classifier was an independent predictor of microalbuminuria. This may help identify high-risk normoalbuminuric patients for preventive strategies for diabetic nephropathy.

Background Diabetic nephropathy (DN) is one of the major late complications of diabetes. Treatment aimed at slowing down the progression of DN is available but methods for early and definitive detection of DN progression are currently lacking. The ‘Proteomic prediction and Renin angiotensin aldosterone system Inhibition prevention Of early diabetic nephRopathy In TYpe 2 diabetic patients with normoalbuminuria trial' (PRIORITY) aims to evaluate the early detection of DN in patients with type 2 diabetes (T2D) using a urinary proteome-based classifier (CKD273). Methods In this ancillary study of the recently initiated PRIORITY trial we aimed to validate for the first time the CKD273 classifier in a multicentre (9 different institutions providing samples from 165 T2D patients) prospective setting. In addition we also investigated the influence of sample containers, age and gender on the CKD273 classifier. Results We observed a high consistency of the CKD273 classification scores across the different centres with areas under the curves ranging from 0.95 to 1.00. The classifier was independent of age (range tested 16-89 years) and gender. Furthermore, the use of different urine storage containers did not affect the classification scores. Analysis of the distribution of the individual peptides of the classifier over the nine different centres showed that fragments of blood-derived and extracellular matrix proteins were the most consistently found. Conclusion We provide for the first time validation of this urinary proteome-based classifier in a multicentre prospective setting and show the suitability of the CKD273 classifier to be used in the PRIORITY trial

Background An improvement in the glomerular filtration rate (GFR) of chronic kidney disease patients has been an underestimated clinical outcome. Although this may be considered as an unexpected disease course, it may provide some insights into possible mechanisms underlying disease remission and/or regression. Therefore, our aim was to identify urinary peptide biomarkers associated with an improvement in estimated GFR (eGFR) over time and to improve patient stratification. Methods Capillary electrophoresis coupled with mass spectrometry (CE-MS) was employed to evaluate the urine peptidome of patients with different types of renal diseases. In total, 376 patients with a slope/year between -1.5% and +1.5% were designated as non-progressors or stable, while 177 patients with a > 5% slope/year were designated as patients with an improved eGFR for state-of-art biomarker discovery and validation. Results We detected 384 significant peptide fragments by comparing the CE-MS data of the stable patients and those with improved renal function in our development cohort. Of these 384, a set of 141 peptides with available amino acid sequence information were used to generate a support vector machine-based classification panel. The biomarker panel was applied to our validation cohort, achieving a moderate area under the curve (AUC) value of 0.85 (81% sensitivity and 81% specificity). The majority of the peptides (78%) from the diagnostic panel arose from different types of collagen. Conclusions We have developed a panel of urinary peptide markers able to discriminate those patients predisposed to improve their kidney function over time and possibly be treated with more specific or less aggressive therapy.

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8-96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing.

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 extracelular matrix (ECM) proteins. In this study, we aimed to investigate the correlation of the es_ESurinary 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, The research presented in this manuscript was supported in part by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No. 642937 (RENALTRACT; MSCA-ITN-2014-642937) and by the “Clinical and system-omics for the identification of the Molecular Determinants of established Chronic Kidney Disease” (iMode-CKD, PEOPLE-ITN-GA-2013-608332). AO was supported by Intensificacion ISCIII and RETIC REDINREN RD016/0009 FEDER funds; MDSN was supported by Miguel Servet program

INTRODUCTION: CKD273 is a urinary biomarker, which in advanced chronic kidney disease predicts further deterioration. We investigated whether CKD273 can also predict a decline of estimated glomerular filtration rate (eGFR) to

We investigate whether the urinary proteome refines the diagnosis of renal dysfunction, which affects over 10% of the adult population. We measured serum creatinine, estimated glomerular filtration rate (eGFR) and 24-h albuminuria in 797 people randomly recruited from a population. We applied capillary electrophoresis coupled with mass spectrometry to measure multi-dimensional urinary proteomic classifiers developed for renal dysfunction (CKD273) or left ventricular dysfunction (HF1 and HF2). Renal function was followed up in 621 participants and the incidence of cardiovascular events in the whole study population. In multivariable-adjusted cross-sectional analyses, higher biomarker levels analysed separately or combined by principal component analysis into a single factor (SF), correlated (P a parts per thousand currency sign 0.010) with worse renal function. Over 4.8 years, higher HF1 and SF predicted (P a parts per thousand currency sign 0.014) lowering of eGFR; higher HF2 predicted (P a parts per thousand currency sign 0.049) increase in serum creatinine and decrease eGFR. HF1, HF2 and SF predicted progression from CKD Stages 2 or a parts per thousand currency sign2 to Stage a parts per thousand yen3, with risk estimates for a 1-SD increment in the urinary biomarkers ranging from 38 to 71% (P a parts per thousand currency sign 0.039). HF1, HF2 and SF yielded a net reclassification improvement of 31-51% (P a parts per thousand currency sign 0.029). Over 6.1 years, 47 cardiovascular events occurred. HF2 and SF, independent of baseline eGFR, 24-h albuminuria and other covariables were significant predictors of cardiovascular complications with risk estimates for 1-SD increases ranging from 32 to 41% (P a parts per thousand currency sign 0.047). The urinary proteome refines the diagnosis of existing or progressing renal dysfunction and predicts cardiovascular complications.

Based on demographic trends, the societies in many developed countries are facing an increasing number and proportion of people over the age of 65. The raise in elderly populations along with improved health-care will be concomitant with an increased prevalence of ageing-associated chronic conditions like cardiovascular, renal, and respiratory diseases, arthritis, dementia, and diabetes mellitus. This is expected to pose unprecedented challenges both for individuals and societies and their health care systems. An ultimate goal of ageing research is therefore the understanding of physiological ageing and the achievement of 'healthy' ageing by decreasing age-related pathologies. However, on a molecular level, ageing is a complex multi-mechanistic process whose contributing factors may vary individually, partly overlap with pathological alterations, and are often poorly understood. Proteome analysis potentially allows modelling of these multifactorial processes. This review summarises recent proteomic research on age-related changes identified in animal models and human studies. We combined this information with pathway analysis to identify molecular mechanisms associated with ageing. We identified some molecular pathways that are affected in most or even all organs and others that are organ-specific. However, appropriately powered studies are needed to confirm these findings based in in silico evaluation.

Background In previous studies, we identified two urinary proteomic classifiers, termed HF1 and HF2, which discriminated subclinical diastolic left ventricular (LV) dysfunction from normal. HF1 and HF2 combine information from 85 and 671 urinary peptides, mainly up- or down-regulated collagen fragments. We sought to validate these classifiers in a population study. Methods In 745 people randomly recruited from a Flemish population (49.8 years; 51.3% women), we measured early and late diastolic peak velocities of mitral inflow (E and A) and mitral annular velocities (e' and a') by conventional and tissue Doppler echocardiography, and the urinary proteome by capillary electrophoresis coupled with mass spectrometry. Results In the analyses adjusted for sex, age, body mass index, blood pressure, heart rate, LV mass index and intake of medications, we expressed effect sizes per 1-SD increment in the classifiers. HF1 was associated with 0.204 cm/s lower e' peak velocity (95% confidence interval, 0.057–0.351; p = 0.007) and 0.145 higher E/e' ratio (0.023–0.268; p = 0.020), while HF2 was associated with a 0.174 higher E/e' ratio (0.046–0.302; p = 0.008). According to published definitions, 67 (9.0%) participants had impaired LV relaxation and 96 (12.9%) had elevated LV filling pressure. The odds of impaired relaxation associated with HF1 was 1.38 (1.01–1.88; p = 0.043) and that of increased LV filling pressure associated with HF2 was 1.38 (1.00–1.90; p = 0.052). Conclusions In a general population, the urinary proteome correlated with diastolic LV dysfunction, proving its utility for early diagnosis of this condition., Highlights • Aim: to validate urinary proteomic markers for LV dysfunction in a population. • Result: LV diastolic dysfunction was associated with the urinary proteome. • Perspective: Screening for subclinical LV dysfunction becomes possible.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in developed nations. Significant albuminuria has long been regarded as the hallmark of DN. Despite this clearly defined indicator, a significant fraction of patients with diabetes develop renal impairment without significant albuminuria, with a variable percentage of patients having advanced kidney disease (1). This observation has resulted in the hypothesis of two distinct pathways (Alb+ and Alb−) of progression toward DN, as suggested by different sets of covariates associated with the two phenotypes. Based on data from the National Health and Nutrition Examination Surveys (NHANES), the number of adults with an estimated glomerular filtration rate (eGFR)

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.