Irene Zubiri

Identification of a urine metabolomic signature in patients with advanced-stage chronic kidney disease

The prevalence of chronic kidney disease (CKD) is increasing and frequently progresses to end-stage renal disease. There is an urgent demand to discover novel markers of disease that allow monitoring disease progression and, eventually, response to treatment. To identify such markers, and as a proof of principle, we determined if a metabolite signature corresponding to CKD can be found in urine. In the discovery stage, we analyzed the urine metabolome by NMR of 15 patients with CKD and compared that with the metabolome of 15 healthy individuals and found a classification pattern clearly indicative of CKD. A validation cohort of urine samples from an additional 16 patients with CKD and 15 controls was then analyzed by (Selected Reaction Monitoring) liquid chromatography-triple quadrupole mass spectrometry and indicated that a group of seven urinary metabolites differed between CKD and non-CKD urine samples. This profile consisted of 5-oxoproline, glutamate, guanidoacetate, α-phenylacetylglutamine, taurine, citrate, and trimethylamine N-oxide. Thus, we identified a panel of urine metabolites differentially present in urine that may help identify and monitor patients with CKD.

A Proteomic Focus on the Alterations Occurring at the Human Atherosclerotic Coronary Intima

Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries.

Metabolomic Profiling for Identification of Novel Potential Biomarkers in Cardiovascular Diseases

Metabolomics involves the identification and quantification of metabolites present in a biological system. Three different approaches can be used: metabolomic fingerprinting, metabolic profiling, and metabolic footprinting, in order to evaluate the clinical course of a disease, patient recovery, changes in response to surgical intervention or pharmacological treatment, as well as other associated features. Characteristic patterns of metabolites can be revealed that broaden our understanding of a particular disorder. In the present paper, common strategies and analytical techniques used in metabolomic studies are reviewed, particularly with reference to the cardiovascular field.

Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis☆

AIMS Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development. METHODS AND RESULTS Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5. CONCLUSION The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer.

UNLABELLED Fatal events derived from coronary atherosclerosis are the major cause of mortality in the developed countries. Proteomic analysis of the atherosclerotic coronary artery has been mainly carried out with whole tissue extracts, making it difficult to distinguish the alterations present in every region of the plaque. For this reason, we have recently described proteins altered in the human coronary intima layer as a consequence of the atherosclerotic disease. In order to complement this work, we aimed here to analyze proteomic alterations occurring within the human coronary media layer. Media layers from human atherosclerotic and preatherosclerotic coronary arteries were isolated by laser microdissection and compared by means of two-dimensional differential in-gel electrophoresis (2D-DIGE). Twelve proteins were found altered, 5 of which were cytoskeleton proteins decreased in the atherosclerotic coronary media. Among these, 4 proteins (filamin A, gelsolin, vinculin and vimentin) were further analyzed by immunohistochemistry and its alteration validated. Such cytoskeleton deregulation evidence, at the molecular level, explains how medial vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. Moreover, an oxidative stress response within the media, leaded by superoxide dismutase 3 and glycolysis activation, may have been triggered by atherosclerosis development. BIOLOGICAL SIGNIFICANCE Although atherosclerosis is mainly a disease of the intima layer, the media plays an important role in the initiation of the pathology, as a source of vascular smooth muscle cells (VSMCs), which migrate into the intima and may additionally be affected by intima layer degeneration through pathogenesis. In fact, intimal thickening has been related to a mechanical compression of the media layer, resulting on a significant thinning of the latter in the atherosclerotic carotid and coronary arteries, which may provoke alterations at a molecular level. Here we provide the first differential proteomic analysis of atherosclerotic coronary media layer, reporting important alterations of this sub-proteome with pathogenesis. It is important to remark a cytoskeleton deregulation observed at the molecular level within VSMCs, which may be explained by a contractile to synthetic phenotype switch. Moreover, atherosclerosis seems to trigger an oxidative stress response within the coronary media layer.

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.

Proteome changes in the myocardium of experimental chronic diabetes and hypertension: role of PPARα in the associated hypertrophy.

Diabetes with or without the presence of hypertension damages the heart. However, there is currently a lack of information about these associated pathologies and the alteration of linked proteins. For these reasons, we were interested in the potential synergistic interaction of diabetes and hypertension in the heart, focusing on the proteome characterization of the pathological phenotypes and the associated hypertrophic response. We treated normotensive and spontaneously hypertensive (SHR) rats with either streptozotocin or vehicle. After 22weeks, type-I diabetic (DM1), SHR, SHR/DM1 and control left-ventricles were studied using proteomic approaches. Proteomics revealed that long-term DM1, SHR and SHR/DM1 rats exhibited 24, 53 and 53 altered proteins in the myocardia, respectively. DM1 myocardium showed over-expression of apoptotic and cytoskeleton proteins, and down-regulation of anti-apoptotic and mitochondrial metabolic enzymes. In both SHR and SHR/DM1 these changes were exacerbated and free fatty-acid (FFA) ß-oxidation enzymes were additionally decreased. Furthermore, SHR/DM1 hearts exhibited a misbalance of specific pro-hypertrophic, anti-apoptotic and mitochondrial ATP-carrier factors, which could cause additional damage. Differential proteins were validated and then clustered into different biological pathways using bioinformatics. These studies suggested the implication of FFA-nuclear receptors and hypertrophic factors in these pathologies. Although key ß-oxidation enzymes were not stimulated in DM1 and hypertensive hearts, peroxisome proliferator-activated receptors-α (PPARα) were potentially activated for other responses. In this regard, PPARα stimulation reduced hypertrophy and pro-hypertrophic factors such as annexin-V in high-glucose and angiotensin-II induced cardiomyocytes. Thus, activation of PPARα could reflect a compensatory response to the metabolic-shifted, apoptotic and hypertrophic status of the hypertensive-diabetic cardiomyopathy.

Tissue proteomics in atherosclerosis: elucidating the molecular mechanisms of cardiovascular diseases

Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehension of the molecular mechanisms can yield very useful information in clinics for prevention, diagnosis and recovery monitoring. Proteomics represents an ideal methodology for this purpose, as proteins constitute the effectors of the different biological processes running during pathogenesis. To date, studies in atherosclerosis have been mainly focused on the search for plasma biomarkers. However, tissue proteomics allows going deeper into tissue secretomes, arterial layers or particular cells of interest, which, in turn, constitutes a more direct approximation to in vivo operating mechanisms. The aim of this review is to report latest advances in tissue proteomics in atherosclerosis and related diseases (e.g., aortic stenosis and ischemic injury).

A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE.

With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub‐proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2‐DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBind™ reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub‐proteome by 2‐DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and well‐resolved 2‐DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano‐LC coupled to an LTQ‐Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated.

KLK1 and ZG16B proteins and arginine–proline metabolism identified as novel targets to monitor atherosclerosis, acute coronary syndrome and recovery

We pursued here the identification of specific signatures of proteins and metabolites in urine which respond to atherosclerosis development, acute event and/or recovery. An animal model (rabbit) of atherosclerosis was developed and molecules responding to atherosclerosis silent development were identified. Those molecules were investigated in human urine from patients suffering an acute coronary syndrome (ACS), at onset and discharge. Kallikrein1 (KLK1) and zymogen granule protein16B (ZG16B) proteins, and l-alanine, l-arabitol, scyllo-inositol, 2-hydroxyphenilacetic acid, 3-hydroxybutyric acid and N-acetylneuraminic acid metabolites were found altered in response to atherosclerosis progression and the acute event, composing a molecular panel related to cardiovascular risk. KLK1 and ZG16B together with 3-hydroxybutyric acid, putrescine and 1-methylhydantoin responded at onset but also showed normalized levels at discharge, constituting a molecular panel to monitor recovery. The observed decreased of KLK1 is in alignment with the protective mechanism of the kallikrein–kinin system. The connection between KLK1 and ZG16B shown by pathway analysis explains reduced levels of toll-like receptor 2 described in atherosclerosis. Metabolomic analysis revealed arginine and proline metabolism, glutathione metabolism and degradation of ketone bodies as the three main pathways altered. In conclusion, two novel urinary panels of proteins and metabolites are here for the first time shown related to atherosclerosis, ACS and patient’s recovery.