Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: Predictors of patient prognosis?

Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients'urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient's recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation.Funding: from Instituto de Salud Carlos III: FIS PI11/01401, PI13/01873, FIS IF08/3667-1, CP09/00229, PI13/00047, PI10/00624, ISCIII-RETIC REDinREN RD012/0021. FEDER funds, Comunidad de Madrid/CIFRA S2010/BMD-2378, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO, IDCSalud (3371/002) and Fundación Conchita Rábago de Jiménez Díaz, Proteomic Facility from Universidad Complutense de Madrid-Fundación Parque Científico de Madrid (UCM-FPCM), Spain, a member of ProteoRed-ISCIII Network member of ProteoRed- ISCIII Networ

Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria

Albuminuria is an indicator of cardiovascular risk and renal damage in hypertensive individuals. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control and prevents development of new-onset-albuminuria. A significant number of patients, however, develop albuminuria despite chronic RAS blockade, and the physiopathological mechanisms are underexplored. Urinary exosomes reflect pathological changes taking place in the kidney. The objective of this work was to examine exosomal protein alterations in hypertensive patients with albuminuria in the presence of chronic RAS suppression, to find novel clues underlying its development. Patients were followed-up for three years and were classified as: a) patients with persistent normoalbuminuria; b) patients developing de novo albuminuria; and c) patients with maintained albuminuria. Exosomal protein alterations between groups were identified by isobaric tag quantitation (iTRAQ). Confirmation was approached by target analysis (SRM). In total, 487 proteins were identified with high confidence. Specifically, 48 proteins showed an altered pattern in response to hypertension and/or albuminuria. Out of them, 21 proteins interact together in three main functional clusters: glycosaminoglycan degradation, coagulation and complement system, and oxidative stress. The identified proteins constitute potential targets for drug development and may help to define therapeutic strategies to evade albuminuria progression in hypertensive patients chronically treated.Instituto de Salud Carlos III, fondos FEDER/FSE (PI11/01401, PI13/01873, PI14/01841, IF08/3667-1, PI11-02239, PI 14/0917, PI11/02432, PI13/01746, PI14/01650, PI16/01334, PT13/0001/0013, CP09/00229, CP15/00129, CPII15/00027), Fundacion SENEFRO, Fundacion Conchita Rabago de Jimenez Diaz, and Redes Tematicas de Investigacion Cooperativa (fondos FEDER/FSE, RD12/0021/0001, RD12/0042/0071). These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP).S

Absence of R-Ras1 and R-Ras2 causes mitochondrial alterations that trigger axonal degeneration in a hypomyelinating disease model

Fast synaptic transmission in vertebrates is critically dependent on myelin for insulation and metabolic support. Myelin is produced by oligodendrocytes (OLs) that maintain multilayered membrane compartments that wrap around axonal fibers. Alterations in myelination can therefore lead to severe pathologies such as multiple sclerosis. Given that hypomyelination disorders have complex etiologies, reproducing clinical symptoms of myelin diseases from a neurological perspective in animal models has been difficult. We recently reported that R-Ras1 and/or R-Ras2 mice, which lack GTPases essential for OL survival and differentiation processes, present different degrees of hypomyelination in the central nervous system with a compounded hypomyelination in double knockout (DKO) mice. Here, we discovered that the loss of R-Ras1 and/or R-Ras2 function is associated with aberrant myelinated axons with increased numbers of mitochondria, and a disrupted mitochondrial respiration that leads to increased reactive oxygen species levels. Consequently, aberrant myelinated axons are thinner with cytoskeletal phosphorylation patterns typical of axonal degeneration processes, characteristic of myelin diseases. Although we observed different levels of hypomyelination in a single mutant mouse, the combined loss of function in DKO mice lead to a compromised axonal integrity, triggering the loss of visual function. Our findings demonstrate that the loss of R-Ras function reproduces several characteristics of hypomyelinating diseases, and we therefore propose that R-Ras1 and R-Ras2 neurological models are valuable approaches for the study of these myelin pathologies.Spanish Ministry of Economy and Competitiveness (RTI2018-096303B-C33) to B. C., (RTI2018-096303B-C31) to F. W., and RTI2018-095166B-I00 to C. G. R. and P. L. and Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) within the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020” (RD16/0008/0020; FIS/PI 18-00754

Analysis of urinary exosomal metabolites identifies cardiovascular risk signatures with added value to urine analysis

Background: Subclinical atherosclerosis may result in fatal cardiovascular (CV) events, but the underlying mechanisms and molecular players leading to disease are not entirely understood. Thus, novel approaches capable of identifying the factors involved in pathological progression and providing a better understanding of the subjacent mechanisms are needed. Extracellular vesicles (EVs) have been shown to have numerous biological functions, and their metabolome has recently generated interest as a source of novel biomarkers. The metabolic content of the exosomes has been so far unexplored in cardiovascular disease (CVD), and here, we developed an analytical strategy aimed at probing urinary exosomal metabolite content and its association to CV risk. Results: Direct analysis of the exosomes without metabolite extraction was evaluated by high-resolution magic angle spinning (1 H HR-MAS). Other two methodologies for the analysis of exosomal metabolites by 1 H NMR were set up, based on methanol or organic solvents sequential extraction. The three methods were compared in terms of the number of detected signals and signal to noise ratio (S/N). The methanol method was applied to identify altered metabolites in the urinary exosomes of subjects with programmed coronary artery by-pass grafting (CABG) versus a control group. Target mass spectrometry (MS) was also performed for differential analysis. The clinical performance of exosomal metabolites of interest in CVD was investigated, and the added value of the exosomes compared to urine analysis was evaluated. Based on S/N ratio, simplicity, reproducibility, and quality of the spectrum, the methanol method was chosen for the study in CVD. A cardiometabolic signature composed by 4-aminohippuric acid, N-1-methylnicotinamide, and citric acid was identified in urinary exosomes. Directly in urine, 4-aminohippuric acid and citric acid do not show variation between groups and changes in N-1-methylnicotinamide are less pronounced, proving the added value of exosomes. Conclusions: We set up a novel methodology to analyze metabolic alterations in urinary exosomes and identified a cardiometabolic signature in these microvesicles. This study constitutes the first evidence of a role for the exosomal metabolism in CVD and demonstrates the possibility to evaluate the urinary exosomal metabolic content by NMR and MS

Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients

Objectives The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. Methods Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. Results Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. Conclusions We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc.EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS 115 56

Design and rationale of a multicentre, randomised, double-blind, placebo-controlled clinical trial to evaluate the effect of vitamin D on ventricular remodelling in patients with anterior myocardial infarction: the VITamin D in Acute Myocardial Infarction (VITDAMI) trial

Introduction:Decreased plasma vitamin D (VD) levels are linked to cardiovascular damage. However, clinical trials have not demonstrated a benefit of VD supplements on left ventricular (LV) remodelling. Anterior ST-elevation acute myocardial infarction (STEMI) is the best human model to study the effect of treatments on LV remodelling. We present a proof-of-concept study that aims to investigate whether VD improves LV remodelling in patients with anterior STEMI. Methods and analysis:The VITamin D in Acute Myocardial Infarction (VITDAMI) trial is a multicentre, randomised, double-blind, placebo-controlled trial. 144 patients with anterior STEMI will be assigned to receive calcifediol 0.266 mg capsules (Hidroferol SGC)/15 days or placebo on a 2:1 basis during 12 months. Primary objective:to evaluate the effect of calcifediol on LV remodelling defined as an increase in LV end-diastolic volume >= 10\% (MRI). Secondary objectives:change in LV end-diastolic and end-systolic volumes, ejection fraction, LV mass, diastolic function, sphericity index and size of fibrotic area; endothelial function; plasma levels of aminoterminal fragment of B-type natriuretic peptide, galectin-3 and monocyte chemoattractant protein-1; levels of calcidiol (VD metabolite) and other components of mineral metabolism (fibroblast growth factor-23 (FGF-23), the soluble form of its receptor klotho, parathormone and phosphate). Differences in the effect of VD will be investigated according to the plasma levels of FGF-23 and klotho. Treatment safety and tolerability will be assessed. This is the first study to evaluate the effect of VD on cardiac remodelling in patients with STEMI. Ethics and dissemination: This trial has been approved by the corresponding Institutional Review Board (IRB) and National Competent Authority (Agencia Espanola de Medicamentos y Productos Sanitarios (AEMPS)). It will be conducted in accordance with good clinical practice (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use-Good Clinical Practice (ICH-GCP)) requirements, ethical principles of the Declaration of Helsinki and national laws. The results will be submitted to indexed medical journals and national and international meetings.The VITDAMI trial is an investigator initiated study, sponsored by the Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz (IIS-FJD). Funding has been obtained from Fondo de Investigaciones Sanitarias (PI14/01567; http://www.isciii.es/) and Spanish Society of Cardiology (http://secardiologia.es/). In addition, the study medication has been provided freely by the pharmaceutical Company FAES FARMA S.A. (Leioa, Vizcaya, Spain; http://faesfarma.com/). This company was the only funder who collaborated in study design (IG-H).S

Immunochip analysis identifies multiple susceptibility loci for systemic sclerosis

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci

AA-amyloidosis in cats (Felis catus) housed in shelters.

Systemic AA-amyloidosis is a protein-misfolding disease characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. A high prevalence of AA-amyloidosis has been reported in captive cheetahs and a horizontal transmission has been proposed. In domestic cats, AA-amyloidosis has been mainly described in predisposed breeds but only rarely reported in domestic short-hair cats. Aims of the study were to determine AA-amyloidosis prevalence in dead shelter cats. Liver, kidney, spleen and bile were collected at death in cats from 3 shelters. AA-amyloidosis was scored. Shedding of amyloid fibrils was investigated with western blot in bile and scored. Descriptive statistics were calculated. In the three shelters investigated, prevalence of AA-amyloidosis was 57.1% (16/28 cats), 73.0% (19/26) and 52.0% (13/25), respectively. In 72.9% of cats (35 in total) three organs were affected concurrently. Histopathology and immunofluorescence of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B = 0.026, CI95% = 0.007-0.046; p = 0.010). AA-amyloidosis was very frequent in shelter cats. Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. In conclusion, AA-amyloidosis was very frequent in shelter cats and those staying longer had more deposits. The cat may represent a natural model of AA-amyloidosis

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file