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The development of de novo albuminuria during chronic renin–angiotensin system (RAS) suppression is a clinical entity that remains poorly recognized in the biomedical literature. It represents a clear increment in global cardiovascular (CV) and renal risk that cannot be counteracted by RAS suppression. Although not specifically considered, it is clear that this entity is present in most published and ongoing trials dealing with the different forms of CV and renal disease. In this review, we focus on the mechanisms promoting albuminuria, and the predictors and new markers of de novo albuminuria, as well as the potential treatment options to counteract the excretion of albumin. The increase in risk that accompanies de novo albuminuria supports the search for early markers and predictors that will allow practising physicians to assess and prevent the development of de novo albuminuria in their patients. Sin financiación 5.237 JCR (2018) Q1, 19/136 Medicine, Research & Experimental 1.946 SJR (2018) Q1, 167/2844 Medicine (miscellaneous) No data IDR 2018 UEM

Acute kidney injury (AKI) is one of the main complications in acute care medicine and a risk factor for chronic kidney disease (CKD). AKI incidence has increased; however, its diagnosis has limitations and physiopathological mechanisms are underexplored. We investigated urine samples, aiming to identify major metabolite changes during human AKI evolution. Metabolic signatures found were further explored for a potential link to severity of injury. Twenty-four control subjects and 38 hospitalized patients with AKI were recruited and urine samples were collected at the time of diagnosis, during follow-up and at discharge. Nuclear magnetic resonance (NMR) was used in a first discovery phase for identifying potential metabolic differences. Target metabolites of interest were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS) in an independent group. Underlying metabolic defects were further explored by kidney transcriptomics of murine toxic AKI. Urinary 2-hydroxybutyric acid, pantothenic acid, and hippuric acid were significantly downregulated and urinary N-acetylneuraminic acid, phosphoethanolamine, and serine were upregulated during AKI. Hippuric acid, phosphoethanolamine, and serine showed further downregulation/upregulation depending on the metabolite in acute tubular necrosis (ATN) AKI compared to prerenal AKI. Kidney transcriptomics disclosed decreased expression of cystathionase, cystathionine-β-synthase, and ethanolamine-phosphate cytidylyltransferase, and increased N-acetylneuraminate synthase as the potentially underlying cause of changes in urinary metabolites. A urinary metabolite panel identified AKI patients and provided insight into intrarenal events. A urine fingerprint made up of six metabolites may be related to pathophysiological changes in oxidative stress, energy generation, and H2S availability associated with AKI.

// Laura Gonzalez-Calero 1 , Paula J. Martinez 1 , Marta Martin-Lorenzo 1 , Montserrat Baldan-Martin 2 , Gema Ruiz-Hurtado 3 , Fernando de la Cuesta 2 , Eva Calvo 4 , Julian Segura 3 , Juan Antonio Lopez 5 , Jesus Vazquez 5 , Maria G. Barderas 2 , Luis M. Ruilope 3 , Fernando Vivanco 1, 6 and Gloria Alvarez-Llamas 1 1 Department of Immunology, IIS-Fundacion Jimenez Diaz, REDinREN, Madrid, Spain 2 Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos SESCAM, Toledo, Spain 3 Hypertension Unit, Instituto de Investigacion Imas12, Hospital Universitario 12 de Octubre, Madrid, Spain 4 Ibermutuamur, Madrid, Spain 5 Laboratory of Cardiovascular Proteomics CNIC, Madrid, Spain 6 Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain Correspondence to: Gloria Alvarez-Llamas, email: galvarez@fjd.es Luis M. Ruilope, email: ruilope@ad-hocbox.com Keywords: exosomes, hypertension, albuminuria, renin-angiotensin system, proteomics Received: March 13, 2017 Accepted: April 20, 2017 Published: May 11, 2017 ABSTRACT 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.

Resumen Introduccion Las vesiculas extracelulares (EV) son liberadas al torrente circulatorio por determinados tipos celulares a consecuencia de procesos de transporte, activacion o muerte celular. El recuento de EV circulantes de origen plaquetario y endotelial ha demostrado tener un importante papel como biomarcador de riesgo cardiovascular. Por lo tanto, el proteoma de estas EV podria reflejar los procesos celulares subyacentes en pacientes con hipertension arterial y albuminuria. Material y metodos El contenido proteico de las EV circulantes se ha analizado mediante cromatografia liquida acoplada a espectrometria de masas. Las EV han sido aisladas mediante un protocolo de ultracentrifugacion optimizado para evitar la contaminacion de proteinas del plasma. La pureza de la fraccion aislada ha sido verificada mediante microscopia electronica y confocal, y por citometria de flujo. Resultados En este estudio mostramos un metodo de gran rendimiento y pureza para obtener extractos proteicos de EV circulantes de pacientes hipertensos con/sin albuminuria para analisis proteomico. Ademas, aportamos el proteoma de referencia de EV de estos pacientes, compuesto por 2.463 proteinas, y demostramos que las proteinas transportadas por estas vesiculas estan relacionadas con procesos cruciales involucrados en el riesgo cardiovascular asociado. Conclusion El proteoma de las EV circulantes constituye una interesante fuente de indicadores para la evaluacion de procesos relacionados con el riesgo cardiovascular en pacientes hipertensos con bloqueo del sistema renina-angiotensina.

Background High albuminuria is a strong predictor of development of cardiovascular events in hypertensive patients. The search for predictors identifying patients at risk of developing high albuminuria or presenting a more rapid progression in this parameter may represent an effective strategy for adequate intervention and better outcome. Methods and results Initially we investigated 24 patients presenting with normoalbuminuria, de novo albuminuria and sustained albuminuria. Plasma proteomics disclosed an upregulation of ceruloplasmin (CP), haptoglobin (HP) and alpha 1-acid glycoprotein (ORM1) that in a second step were selected for validation using turbidimetry assay in a cohort of 105 subjects. The validation showed that HP and ORM1 proteins were increased in patients presenting with very high albuminuria and potential irreversible kidney damage. CP and HP correlated positively with albuminuria values in normoalbuminuric patients. Finally, the levels of ORM1 and CP were increased in patients who progressed in their levels of albuminuria. Conclusions Our findings show that these proteins may potentially be useful for predicting the development of high albuminuria and to monitor renal damage.

Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. The molecular anatomy of healthy and atherosclerotic tissue is pursued to identify ongoing molecular changes in atherosclerosis development. Mass Spectrometry Imaging (MSI) accounts with the unique advantage of analyzing proteins and metabolites (lipids) while preserving their original localization; thus two dimensional maps can be obtained. Main molecular alterations were investigated in a rabbit model in response to early development of atherosclerosis. Aortic arterial layers (intima and media) and calcified regions were investigated in detail by MALDI-MSI and proteins and lipids specifically defining those areas of interest were identified. These data further complement main findings previously published in J Proteomics (M. Martin-Lorenzo et al., J. Proteomics. (In press); M. Martin-Lorenzo et al., J. Proteomics 108 (2014) 465–468.) [1,2].

The molecular anatomy of healthy and atherosclerotic tissue is pursued here to identify ongoing molecular changes in atherosclerosis development. Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. Mass spectrometry imaging (MSI) is a novel unexplored ex vivo imaging approach in CVD able to provide in-tissue molecular maps. A rabbit model of early atherosclerosis was developed and high-spatial-resolution MALDI-MSI was applied to comparatively analyze histologically-based arterial regions of interest from control and early atherosclerotic aortas. Specific protocols were applied to identify lipids and proteins significantly altered in response to atherosclerosis. Observed protein alterations were confirmed by immunohistochemistry in rabbit tissue, and additionally in human aortas. Molecular features specifically defining different arterial regions were identified. Localized in the intima, increased expression of SFA and lysolipids and intimal spatial organization showing accumulation of PI, PG and SM point to endothelial dysfunction and triggered inflammatory response. TG, PA, SM and PE-Cer were identified specifically located in calcified regions. Thymosin β4 (TMSB4X) protein was upregulated in intima versus media layer and also in response to atherosclerosis. This overexpression and localization was confirmed in human aortas. In conclusion, molecular histology by MS Imaging identifies spatial organization of arterial tissue in response to atherosclerosis.

Albuminuria development in hypertensive patients is an indicator of higher cardiovascular (CV) risk and renal damage. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control but it does not prevent from albuminuria development. We pursued the identification of protein indicators in urine behind albuminuria development in hypertensive patients under RAS suppression. Urine was collected from 100 patients classified in three groups according to albuminuria development: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Quantitative analysis was performed in a first discovery cohort by isobaric labeling methodology. Alterations of proteins of interest were confirmed by target mass spectrometry analysis in an independent cohort. A total of 2416 proteins and 1223 functional categories (coordinated protein responses) were identified. Immune response, adhesion of immune and blood cells, and phagocytosis were found significantly altered in patients with albuminuria compared to normoalbuminuric individuals. The complement system C3 increases, while Annexin A1, CD44, S100A8 and S100A9 proteins showed significant diminishment in their urinary levels when albuminuria is present. This study reveals specific links between immune response and controlled hypertension in patients who develop albuminuria, pointing to potential protein targets for novel and future therapeutic interventions. The authors acknowledge Lucia Guerrero and Maria Cruz Casal from Hospital 12 de Octubre for their participation in samples collection. Instituto de Salud Carlos III co- supported by FEDER grants (PI11/01401, PI13/01873, PI14/01650, PI16/01334, CPII15/00027, PT13/0001/0013, IF08/3667-1, PI11/02239, PI11/02432, PI14/0917, PI14/01841, PIE13/00045, CP15/00129, PI13/01746, REDinREN (RD12/0021/0001, RD12/0042/0071 and RD16/0009)), and Fundacion SENEFRO. These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP). Sí

Acute kidney injury (AKI) is one of the main complications in acute care medicine and a risk factor for chronic kidney disease (CKD). AKI incidence has increased; however, its diagnosis has limitations and physiopathological mechanisms are underexplored. We investigated urine samples, aiming to identify major metabolite changes during human AKI evolution. Metabolic signatures found were further explored for a potential link to severity of injury. Twenty-four control subjects and 38 hospitalized patients with AKI were recruited and urine samples were collected at the time of diagnosis, during follow-up and at discharge. Nuclear magnetic resonance (NMR) was used in a first discovery phase for identifying potential metabolic differences. Target metabolites of interest were confirmed by liquid chromatography-mass spectrometry (LC-MS/MS) in an independent group. Underlying metabolic defects were further explored by kidney transcriptomics of murine toxic AKI. Urinary 2-hydroxybutyric acid, pantothenic acid, and hippuric acid were significantly downregulated and urinary N-acetylneuraminic acid, phosphoethanolamine, and serine were upregulated during AKI. Hippuric acid, phosphoethanolamine, and serine showed further downregulation/upregulation depending on the metabolite in acute tubular necrosis (ATN) AKI compared to prerenal AKI. Kidney transcriptomics disclosed decreased expression of cystathionase, cystathionine-β-synthase, and ethanolamine-phosphate cytidylyltransferase, and increased N-acetylneuraminate synthase as the potentially underlying cause of changes in urinary metabolites. A urinary metabolite panel identified AKI patients and provided insight into intrarenal events. A urine fingerprint made up of six metabolites may be related to pathophysiological changes in oxidative stress, energy generation, and HThe urinary metabolome reflects AKI evolution and severity of injury. Kidney transcriptomics revealed enzymatic expression changes. Enzymatic expression changes may be the potentially underlying cause of changes in urine metabolites. Identified metabolite changes link oxidative stress, energy generation, and H

Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (> 30 mg/L albumin in urine) renal disease.

// Fernando de la Cuesta 1, 6 , Montserrat Baldan-Martin 1 , Rafael Moreno-Luna 1 , Gloria Alvarez-Llamas 2 , Laura Gonzalez-Calero 2 , Laura Mourino-Alvarez 1 , Tamara Sastre-Oliva 1 , Juan A. Lopez 3 , Jesus Vazquez 3 , Gema Ruiz-Hurtado 4 , Julian Segura 4 , Fernando Vivanco 2, 5 , Luis M. Ruilope 4 , Maria G. Barderas 1 1 Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos (HNP), SESCAM, Toledo, Spain 2 Department of Immunology, IIS-Fundacion Jimenez Diaz, Madrid, Spain 3 Unidad de Proteomica CNIC, Madrid, Spain 4 Unidad de Hipertension, Instituto de Investigacion i + 12, Hospital Universitario 12 de Octubre, Madrid, Spain 5 Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense, Madrid, Spain 6 Current address: Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK Correspondence to: Maria G. Barderas, email: megonzalezb@sescam.jccm.es Luis M. Ruilope, email: ruilope@ad-hocbox.com Keywords: extracellular vesicles, proteomics, endothelial dysfunction, hypertension, albuminuria Received: August 17, 2016 Accepted: December 05, 2016 Published: February 01, 2017 ABSTRACT Despite of the great advances in anti-hypertensive therapies, many patients under Renin-Angiotensin- System (RAS) suppression develop albuminuria, which is a clear indicator of therapeutic inefficiency. Hence, indicators of vascular function are needed to assess patients’ condition and help deciding future therapies. Proteomic analysis of circulating extracellular vesicles (EVs) showed two proteins, kalirin and chromodomain-helicase-DNA-binding protein 7 (CHD7), increased in albuminuric patients. A positive correlation of both with the expression of the endothelial activation marker E-selectin was found in EVs. In vitro analysis using TNFα-treated adult human endothelial cells proved their involvement in endothelial cell activation. Hence, we propose protein levels of kalirin and CHD7 in circulating EVs as novel endothelial dysfunction markers to monitor vascular condition in hypertensive patients with albuminuria.

Background The most common valve diseases are calcific aortic stenosis (AS) and aortic regurgitation (AR). The former is characterized by thickening of valve leaflets followed by progressive calcification, which produces progressive aortic valve (AV) narrowing, increased pressure afterload on the left ventricle (LV) and subsequent LV hypertrophy. On the other hand, AR is due to malcoaptation of the valve leaflets with resultant diastolic reflux of blood from aorta back to the LV producing volume and pressure overload and progressive LV dilatation. In order to isolate the molecular mechanisms taking place during AS, we have used an integrated "–omic" approach to compare plasma samples from AS and from AR patients used as controls. The final purpose of this work is to find molecular changes in response to the calcification of the AV, diminishing the effects of the AV dysfunction. Methods and results Using two-dimensional difference gel electrophoresis (2D-DIGE) and gas chromatography coupled to mass spectrometry (GC-MS) in a cohort of 6 subjects, we have found differences in 24 protein spots and 19 metabolites, respectively. Among them, 7 proteins and 3 metabolites have been verificated by orthogonal techniques (SRM or turbidimetry): fibrinogen beta and gamma chain, vitronectin, apolipoprotein C-II, antithrombin III, haptoglobin, succinic acid, pyroglutamic acid and alanine. Classification according to their main function showed alterations related to coagulation, inflammation, oxidative stress, response to ischemia and lipid metabolism, defining 4 different molecular panels that characterize AS with high specificity and sensitivity. Conclusion These results may facilitate management of these patients by making faster diagnostics of the disease and better understand these pathways for regulating its progression.

Albuminuria is a risk factor strongly associated with cardiovascular disease, the first cause of death in the general population. It is well established that renin–angiotensin system suppressors prevent the development of new-onset albuminuria in naïf hypertensive patients and diminish its excretion, but we cannot forget the percentage of hypertensive patients who develop de novo albuminuria. Here, we applied multiple proteomic strategy with the purpose to elucidate specific molecular pathways involved in the pathogenesis and provide predictors and chronic organ damage indicators. Briefly, 1143 patients were followed up for a minimum period of 3 years. One hundred and twenty-nine hypertensive patients chronically renin–angiotensin system suppressed were recruited, classified in 3 different groups depending on their albuminuria levels (normoalbuminuria, de novo albuminuria, and sustained albuminuria), and investigated by multiple proteomic strategies. Our strategy allowed us to perform one of the deepest plasma proteomic analysis to date, which has shown 2 proteomic signatures: (1) with predictive value of de novo albuminuria and (2) sustained albuminuria indicator proteins. These proteins are involved in inflammation, immune as well as in the proteasome activation occurring in situations of endoplasmic reticulum stress. Furthermore, these results open the possibility of a future strategy based on anti-immune therapy to treat hypertension which could help to prevent the development of albuminuria and, hence, the progression of kidney damage.

Background Hypertension is a multi-factorial disease of increasing prevalence and a major risk factor for cardiovascular mortality even in the presence of adequate treatment. Progression of cardiovascular disease (CVD) occurs frequently during chronic renin-angiotensin-system (RAS) suppression, and albuminuria is a marker of CV risk. High prevalence of albuminuria in treated hypertensive patients has been demonstrated, but there are no available markers able to predict evolution. The aim of this study was the identification of novel indicators of albuminuria progression measurable in urine of diabetic and non-diabetic patients. Methods 1143 hypertensive patients under chronic treatment were followed for a minimum period of 3 years. Among them, 105 diabetic and non-diabetic patients were selected and classified in three groups according to albuminuria development during follow-up: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Differential urine analysis was performed by 2D gel electrophoresis (2D-DIGE) and further confirmed by liquid chromatography-mass spectrometry. Non-parametric statistical tests were applied. Results CD59 glycoprotein and alpha-1 antitrypsin (AAT) resulted already altered in patients developing albuminuria de novo, with a similar response in those with maintained albuminuria. A prospective study in a sub-group of normoalbuminuric patients who were clinically followed up for at least 1 year from urine sampling, revealed CD59 and AAT proteins significantly varied in the urine collected from normoalbuminurics who will negatively progress, serving as predictors of future albuminuria development. Conclusions CD59 and AAT proteins are significantly altered in hypertensive patients developing albuminuria. Interestingly, CD59 and AAT are able to predict, in normoalbuminuric individuals, who will develop albuminuria in the future, being potential predictors of vascular damage and CV risk. These findings contribute to early identify patients at risk of developing albuminuria even when this classical predictor is still in the normal range, constituting a novel strategy towards a prompt and more efficient therapeutic intervention with better outcome. Electronic supplementary material The online version of this article (doi:10.1186/s12933-016-0331-7) contains supplementary material, which is available to authorized users.

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 Network

Background Cardiovascular disease (CVD) is the leading cause of death globally, being atherosclerosis the main cause. Main risk factors are known and current effort is very much dedicated to improve prevention. However, the asymptomatic and silent course of atherosclerosis hampers an accurate and individualized risk evaluation. Objectives Here we investigate subjacent molecular changes taking place in arterial tissue which can be ultimately translated in a measurable fingerprint in plasma. Methods First, we applied a combined approach to find out main molecular alterations at protein and metabolite level in response to early atherosclerosis development in a rabbit model. A potential reflection of all these alterations observed in aortic tissue was investigated in rabbit plasma and further analyzed in a translational study in human plasma from 62 individuals. Results Data link the structural remodeling taking place in atherosclerotic arteries in terms of loss of contractile properties and favored cellular migration, with an up-regulation of integrin linked kinase, tropomyosin isoform 2 and capping protein gelsolin-like, and a down-regulation of vinculin. A molecular response to oxidative stress is evidenced, involving changes in the glucose metabolism enzymes pyruvate kinase (PKM) and phosphoglycerate kinase (PGK), and pyruvate. Up-regulation of aspartate connects different changes observed in amino acid metabolism and, additionally, alterations in the phosphatidylcholine route of the glycerophospholipid metabolism were found. Conclusions A specific molecular marker panel composed by PKM, valine and pyruvate is shown here linked to cardiovascular risk.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease. DN progresses silently and without clinical symptoms at early stages. Current noninvasive available markers as albuminuria account with severe limitations (late response, unpredictable prognosis, and limited sensitivity). Thus, it urges the discovery of novel markers to help in diagnosis and outcome prediction. Tissue proteomics allows zooming-in where pathophysiological changes are taking place. We performed a differential analysis of renal tissue proteome in a rat model of early DN by 2-dimensional differential gel electrophoresis and mass spectrometry. Confirmation was performed by Western blot, immunohistochemistry (IHC), and selected reaction monitoring (SRM). Rat urine samples were collected and exosomes were isolated from urine to evaluate if these microvesicles reflect changes directly occurring at tissue level. The protein showing maximum altered expression in rat tissue in response to DN was further analyzed in human kidney tissue and urinary exosomes. Regucalcin protein or senescence marker protein-30 (SMP30) (Swiss-Prot Q03336) was found to be strongly downregulated in DN kidney tissue compared with healthy controls. The same trend was observed in exosomes isolated from urine of control and DN rats. These data were further confirmed in a pilot study with human samples. IHC revealed a significant decrease of regucalcin in human kidney disease tissue vs control kidney tissue, and regucalcin was detected in exosomes isolated from healthy donors' urine but not from kidney disease patients. In conclusion, regucalcin protein expression is reduced in DN kidney tissue and this significant change is reflected in exosomes isolated from urine. Urinary exosomal regucalcin represents a novel tool, which should be explored for early diagnosis and progression monitoring of diabetic kidney disease.

Exosomes have proven roles in regulating immune response, antigen presentation, RNA and protein transfer, and cell–cell (organ–organ) interaction/signaling. These microvesicles can be considered a mechanism of non-classical secretion of proteins, and they represent a sub-proteome, thus assisting in the difficult task of biomarker discovery in a biological fluid as urine, plasma or serum. A potential role of exosomes in the cardio-renal syndrome is currently underexplored. Cardiovascular disease (CVD) continues to be the leading cause of morbidity and mortality worldwide and, particularly, rates of cardiovascular events and death consistently increase as kidney function worsens. In other words, chronic kidney disease acts as a risk multiplier. Unfortunately, the relationship between markers of cardiovascular risk in kidney pathology often differs from that in the general population. Efforts in the search for novel action mechanisms simultaneously operating in both pathologies are thus of maximum interest. This article focuses to the role of exosomes in cardiovascular and renal diseases, in the search for novel key targets of interaction between heart and kidneys.

Urine is a source of potential markers of disease. In the context of renal disease, urine is particularly important as it may directly reflect kidney injury. Current markers of renal dysfunction lack both optimal specificity and sensitivity, and improved technologies and approaches are needed. There is no clear consensus about the best sample pretreatment procedure for 2DE analysis of the urine proteome. Sample pretreatment conditions spots resolution and detection sensitivity, critically. As a first goal, we exhaustively compared eight different sample cleaning and protein purification methodologies for 2DE analysis of urine from healthy individuals. Oasis® HLB cartridges allowed the detection of the highest number of low molecular weight proteins; while PD10 desalting columns resulted in the highest number of detected spots in the high molecular weight area. Sample pretreatment strategies were also explored in the context of proteinuria, a clinical condition often associated to renal damage. Testing of urine samples from 13 patients with hypertension or kidney disease and different levels of proteinuria identified Oasis® HLB cartridge purification in combination with albumin depletion by ProteoPrep kit as the best option for urine proteome profiling from patients with proteinuric (30 mg/L albumin in urine) renal disease.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), the most frequent cause of end-stage renal disease (ESRD). Exosomes isolated from urine are considered a rich non-invasive source of markers for renal events. Proteinuria associated with DN patients at advanced stages may result in “contamination” of exosomal fraction by co-precipitation of high abundance urine proteins, making it enormously difficult to obtain a reliable comparison of healthy individuals and DN patients and to detect minor proteins. We evaluated different protocols for urinary exosome isolation (ultracentrifugation-based and Exoquick® reagent-based) in combination with an easy and quick depletion procedure of contaminating high abundance proteins (albumin). The optimal methodology was then applied to investigate the proteome of human urinary exosomes in DN and controls using spectral counting LC–MS/MS analysis followed by selected reaction monitoring (SRM) confirmation. A panel of 3 proteins (AMBP, MLL3, and VDAC1) is differentially present in urinary exosomes from DN patients, opening a new field of research focused on improving diagnosis and follow-up of this pathology. Biological significance Diabetic nephropathy (DN) is a progressive proteinuric kidney disease, a major complication of diabetes mellitus, and the most frequent cause of end-stage renal disease. Current markers of disease (i.e. creatinine and urinary albumin excretion) have proven limitations (i.e. some patients regress to normoalbuminuria, kidney damage may be already present in recently diagnoses microalbuminuric patients and renal function may decrease in the absence of significant albuminuria). We show here the first study on human DN proteome of urinary exosomes. Proteinuria associated to DN patients resulting in contamination of exosomal fraction and the associated difficulty to reliably compare healthy and disease conditions, are here overcome. A combined methodology pointed to increase exosomal proteome recovery and depletion of high-abundance proteome was here set-up. A total of 352 proteins were here identified for the first time associated to human urinary exosomes. Label-free quantitative comparison of DN urinary exosomes vs control group and SRM further validation, resulted in the discovery of a panel of three proteins (AMBP, MLL3 and VDAC1) which changes in DN, opening a new field of research focused to improve diagnosis and follow-up of this pathology.

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.

Purpose: Search of progression and recovery markers in atherosclerosis disease. Methods: An animal model of early atherosclerosis was done in rabbits fed with different diets, regular diet and cholesterol-rich (control and pathological group). Urine samples from animals were analyzed in the search for alterations of the proteome and metabolome (by 2DE-DIGE and NMR, respectively). Confirmation analyses were performed by SRM-(LC-MS/MS). In a pilot translational study, human urine samples from healthy donors and NSTEACS patients at the onset and discharge were analyzed. Results: Four proteins were found significant altered between the two animal groups. Cathepsin D is increased in urine from pathological animals and Hemopexin, Kallikrein 1 (KLK1) and Zymogen granule protein 16B (ZG16B) are decreased. After translational studies KLK1 and ZG16B are proposed to be markers of both, progression and recovery. NMR pops up twenty metabolites significantly varied at early stages of atherosclerosis between animal groups. Eight metabolites increased in urine from pathological animals and twelve metabolites showed the opposite trend (decreased). Two panels of six and three metabolites are found as responders to atherosclerosis progression or recovery from an acute event, respectively. Metabolism changes of particular sugars, hydroxy acids, amino acids, cyclic alcohols, polyamines and imidazolidines are here shown to be associated to atherosclerosis. Conclusions: These data confirm that omics approaches are extremely valuable for the diagnosis and monitoring of atherosclerotic pathologies. Four proteins and twenty metabolites are significantly altered in an early animal model of atherosclerosis. In human samples, two novel urinary panels of proteins and metabolites are here first shown able to monitor atherosclerosis progression and recovery from an acute event.

A characteristic proteomic and metabolomic profile with utility in predicting evolution of patients with arterial hypertension Montserrat Baldan-Martin, Laura Mourino-Alvarez, Fernando De la Cuesta, Laura Gonzalez-Calero, Gloria Alvarez-Llamas, Julian Segura, Cesar Cerezo, Luis R. Padial, Fernando Vivanco, Luis M. Ruilope, Maria G. Barderas. Hospital 12 de Octubre, Madrid, Spain; Hospital Nacional de Paraplejicos, Toledo, Spain; Hospital Virgen de la Salud, Toledo, Spain; IIS-Fundacion Jimenez Diaz, Madrid, Spain; IIS-Hospital 12 de Octubre, Madrid, Spain

Hypertension (HTN) is increasing in prevalence, and albuminuria is a strong indicator of cardiovascular risk and renal damage progression. Despite blood pressure control with chronic treatment, a relevant subgroup of patients develop albuminuria. However, the biological factors responsible for albuminuria development and progression are underexplored. We aimed to identify key metabolic targets and biological pathways involved in the negative progression of cardiovascular and renal damage in hypertensives undergoing chronic treatment. A series of 1533 patients were followed for 5 years to investigate the evolution of albuminuria. Patients were classified as: (1) patients with persistent normoalbuminuria; (2) patients developing de novo albuminuria; and (3) patients with maintained albuminuria. At the end of follow-up, urine from 30 nonhypertensive subjects (control group) and a representative cohort of 118 patients was collected for metabolomic analysis. Metabolic patterns of interest were identified in a first discovery phase by nuclear magnetic resonance and further confirmed by liquid chromatography-mass spectrometry. Metabolites corresponding to HTN or albuminuria were measured in a prospective study carried out in 35 individuals still in normoalbuminuria, to evaluate their potential as predictors of albuminuria development. Nine metabolites were significantly altered, linking β-alanine metabolism, arginine and proline metabolism, and tricarboxylic acid cycle. The prospective study revealed a panel composed of guanidinoacetate, glutamate, and pantothenate, which was able to predict development of albuminuria. These metabolic signatures open new possibilities in hypertensive therapy and cardiovascular risk control, providing prompt and more efficient intervention, particularly in patients with worse cardiovascular prognosis.