Search

Additional file 1: Supplementary Figure S1: S100A9 expression after epithelial injury. A. Urinary calprotectin (S100A8/A9) abundance 4 hours after cardiac bypass-surgery. B–F. Blood urea nitrogen (B), mRNA S100a9 (C), S100A9 immunostaining (D-E) and mRNA Kim1 (F) in sham mice and after bilateral renal ischemia/reperfusion (hours 6, 24 and 48). G–J. mRNA expression of S100a9 and Kim1 in MCT cells submitted to interleukin-1b (IL1β, 10 ng/mL) or tumor necrosis factor-1a (TNFa, 10 ng/mL) (G-H) or hypoxia (I-J). AKI, acute kidney injury; BUN, blood urea nitrogen; Norm, normoxia; Hyp, hypoxia. Supplementary Figure S2: Performances of the peptide-based signature to identify AKI that developed within the first 2 days following cardiac surgery. ROC curves with corresponding AUROC and 95% confidence intervals of the local clinical score (blue, pointed), the full 204 peptides-based score (red), the urinary NGAL level (yellow, pointed) and the nephrocheck ([IGFBP7].[TIMP2] product) in the validation cohort. Supplementary Figure S3: Reduction and combination of the peptide-based signature. A. ROC curves with corresponding AUROC and 95% confidence intervals of the local clinical score (blue, pointed), the full 204 peptides-based score (red), the reduced 17 peptides-based score (black, dashed) and the combination of local clinical and full peptide-based score in the validation cohort. B. List of peptides included in the reduced 17-peptides signature according to their parental protein. LMAN2, Lectin mannose binding 2 ; MGP, Matrix gla protein. Supplementary Figure S4: Performances of the 204 peptides-based signature and the reference urinary biomarker NGAL for AKI prediction in the external ICU validation cohort. A. ROC curves with corresponding AUROC and 95% confidence intervals of the 204 peptides-based score and the reference urinary biomarker NGAL to predict AKI after ICU admission. B. ROC curves with corresponding AUROC and 95% confidence intervals of the 204 peptides-based score and reference urinary biomarker NGAL to predict the development of AKI within seven days after admission. Supplementary Figure S5: Performances of the 204 peptides-based score for in-hospital mortality prediction. Odds-ratio (OR) of in-hospital mortality were calculated with unadjusted, Euroscore-II-adjusted or propensity score-adjusted logistic regression. Supplementary Table S1: Correlations between clinical characteristics and the 204-peptides-based score. BMI, body mass index; PAOD, peripheral artery obliterans disease; COPD, chronic obstructive pulmonary disease; LVEF, left ventricular ejection fraction; eGFR, estimated glomerular filtration rate; CBP, cardiac bypass; RBC, red blood cells. Supplementary Table S2: Performance of the peptide-based score to predict acute kidney injury in the external ICU validation cohort, according to the cause of admission to the intensive care unit. ICU, intensive care unit; AUROC, area under the receiver operating characteristics curve. Supplementary file S1: Methodology. Urinary peptidomics and statistical analyses.

Background and Aims Vascular calcification, leading to aortic stiffening and heart failure, is decisive risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). Promoted by bone mineral disorder and systemic inflammation in CKD patients, vascular calcification is a complex mechanism involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. Despite intensive research efforts in recent years, available treatments have limited effect and none of them prevent or reverse vascular calcification. The aim of this study was to analyse the serum proteome of CKD stage 3-4 patients in order to unravel new molecular changes associated to CV morbid-mortality and to decipher the role of novel candidates on vascular calcification to provide potential new therapeutic agents. Method In this study we used serum samples from two independent cohorts: 112 CKD stage 3-4 patients with a 4 years follow-up for CV events and 222 CKD stage 5 patients exhibiting a broad range of calcification degree determined by histological quantification in the epigastric and/or iliac artery. Serum proteome analysis was performed using tandem mass-spectrometry in a subcohort of 66 CKD3-4 patients and validation of protein candidates was performed using ELISA in the two full cohorts. Human primary vascular smooth muscle cells and mouse aortic rings were used for calcification assays. Calcium content was quantified using QuantiChrom calcium assay kit and calcium deposition was visualized by Alizarin Red and Von Kossa staining. Results Among 443 proteins detected in the serum of CKD3-4 patients, 134 displayed significant modified abundance in patients with CV events (n=32) compared to patients without (n=34). One of the most prominent changes was increased level of calprotectin (up to 8.6 fold, P Conclusion Circulating calprotectin is a novel predictor of CV outcome and mortality in CKD patients. Calprotectin also shows calcification-inducing properties and its blockade by paquinimod alleviates its effects. Future experiments will consist in deciphering the signalling pathways involved in the regulation of calcification by calprotectin and evaluating in vivo the therapeutic potential of paquinimod on the development of medial vascular calcification lesions associated with CKD.

Introduction La calcification vasculaire associee a la maladie renale chronique (MRC) est un facteur de risque decisif de complications cardiovasculaires (CCV). Pourtant, les options therapeutiques restent limitees. La calprotectine est une alarmine jouant un role important dans diverses maladies auto-immunes et inflammatoires. Description L’objectif de ce travail etait d’etudier le role de la calprotectine et d’evaluer le potentiel therapeutique du paquinimod, un medicament inhibant la calprotectine, dans la calcification vasculaire associee a la MRC. Methodes La concentration de calprotectine circulante a ete mesuree par ELISA chez 170 patients MRC avec ou sans calcification vasculaire, ainsi que chez 283 patients MRC stades 3–4 ou en dialyse ayant developpe ou non des CCV au cours du suivi. L’effet de la calprotectine et du paquinimod sur la calcification a ete mesure dans des cellules primaires musculaires lisses vasculaires (CMLV) humaines et dans des anneaux d’aorte murins. L’effet du paquinimod sur la calcification a ete egalement evalue dans un modele animal de MRC (nephrectomie subtotale) avec calcifications vasculaires. Resultats Les taux circulant de calprotectine sont augmentes chez les patients MRC avec calcification par comparaison aux patients sans calcification. De plus, une augmentation de la calprotectine circulante est significativement et independamment associee a la survenue de CCV. Le traitement par la calprotectine de CMLV et d’anneaux d’aorte induit une augmentation significative de la calcification via l’activation de TLR4 et de RAGE et cet effet est significativement inhibe par le paquinimod. Enfin, le traitement par le paquinimod reduit le processus de calcification vasculaire chez la souris en cas d’atteinte renale. Conclusion Ces resultats suggerent que la calprotectine est associee aux CCV chez le patient MRC avec une implication en particulier dans le processus de calcification vasculaire. Ainsi, le ciblage de la calprotectine par le paquinimod pourrait etre une approche therapeutique innovante et efficace afin d’ameliorer la survie de ces patients.

Introduction La calcification vasculaire associee a la maladie renale chronique (MRC) est un facteur de risque decisif de complications cardiovasculaires (CCV). Pourtant, les options therapeutiques ciblant ce processus restent limitees. La calprotectine est une alarmine pouvant activer les recepteurs TLR4 et RAGE et jouant un role important dans diverses maladies auto-immunes et inflammatoires. Description L’objectif de ce travail etait d’etudier le role de la calprotectine en tant que nouvel acteur de la calcification vasculaire associee a la MRC et d’evaluer le potentiel therapeutique du paquinimod, un medicament inhibant la calprotectine. Methodes La concentration de calprotectine circulante a ete mesuree par ELISA dans : (i) le serum de 63 patients MRC (stades 3 et 4) ayant developpe (n = 34) ou non (n = 29) des CCV au cours du suivi sur 4 ans et (ii) dans un modele animal de MRC (nephrectomie 5/6) avec calcifications vasculaires induites par un regime riche en phosphate. Le contenu en calcium (marqueur de calcification) a ete mesure dans des cellules primaires musculaires lisses vasculaires (CMLV) humaines et dans des anneaux d’aorte murins traites par la calprotectine avec ou sans pre-traitement par le paquinimod. Resultats Les taux de calprotectine serique sont augmentes chez les patients MRC avec CCV par comparaison aux patients sans CCV. De plus, la concentration plasmatique de calprotectine est augmentee chez la souris en cas d’atteinte renale avec calcifications vasculaires. Le traitement par la calprotectine de CMLV et d’anneaux d’aorte induit une augmentation significative de la calcification et cet effet est significativement inhibe par le paquinimod. Conclusion Ces resultats suggerent que la calprotectine est associee aux CCV chez le patient MRC avec une implication en particulier dans le processus de calcification vasculaire. Ainsi, le ciblage de la calprotectine par le paquinimod pourrait etre une approche therapeutique innovante et efficace afin d’ameliorer la survie de ces patients.

Fibroblast activation protein-alpha (FAP') is a membrane protein with type I collagenase activity expressed during embryogenesis. Reaching adolescence, FAP' expression decreases, only increasing during wound healing, tumour growth or myocardial infarction. FAP' expression was recently observed in bone-marrow mesenchymal stem cells (MSCs) and ongoing research from our group revealed its presence on MSCs from the dental tissue. In this project, we hypothesized that FAP' plays an important role in dental root development. For that we aimed to study FAP' expression in dental stem cells involved in root development; dental follicle stem cells (DFSCs) and Hertwig's Epithelial Root Sheath (HERS), a bilayer of epithelial cells, and Stem Cells from the Apical Papilla (SCAPs), providing information about this tissue. The expression of FAP' was also studied in ameloblastoma, a tumour provoked during tooth development. To achieve this, a characterization through immunohistochemistry and immunofluorescence analysis was performed. We report the presence of FAP' on DFSCs and SCAPs. We also provided insights in the apical papilla identifying two regions: cortex fibrosa, at the periphery, and medulla, in the core, both positive for FAP'. TGF-'1 and SDF-1 were found in the cortex fibrosa co-located with FAP' and SCAPs showed enhanced migration in response to SDF-1. FAP' was also identified in ameloblastoma. These results suggest a role of FAP' in the migration of dental stem cells during root maturation related to its collagenase function.