Cardiomyocyte-specific deletion of GCN5L1 reduces lysine acetylation and attenuates diastolic dysfunction in aged mice by improving cardiac fatty acid oxidation.

Cardiac mitochondrial dysfunction is a critical contributor to the pathogenesis of aging and many age-related conditions. As such, complete control of mitochondrial function is critical to maintain cardiac efficiency in the aged heart. Lysine acetylation is a reversible post-translational modification shown to regulate several mitochondrial metabolic and biochemical processes. In the present study, we investigated how mitochondrial lysine acetylation regulates fatty acid oxidation (FAO) and cardiac function in the aged heart. We found a significant increase in mitochondrial protein acetylation in the aged heart which correlated with increased level of mitochondrial acetyltransferase-related protein GCN5L1. We showed that acetylation status of several fatty acid and glucose oxidation enzymes (long-chain acyl-coenzyme A dehydrogenase, hydroxyacyl-coA dehydrogenase, and pyruvate dehydrogenase) were significantly up-regulated in aged heart which correlated with decreased enzymatic activities. Using a cardiac-specific GCN5L1 knockout (KO) animal model, we showed that overall acetylation of mitochondrial proteins was decreased in aged KO animals, including FAO proteins which led to improved FAO activity and attenuated cardiac diastolic dysfunction observed in the aged heart. Together, these findings indicate that lysine acetylation regulates FAO in the aged heart which results in improved cardiac diastolic function and this is in part regulated by GCN5L1.

Identifying a urinary peptidomics profile for hypertension in young adults: The African-PREDICT study: Urinary peptidomics and hypertension: Urinary peptidomics and hypertension.

Hypertension is one of the most important and complex risk factors for cardiovascular diseases (CVDs). By using urinary peptidomics analyses, we aimed to identify peptides associated with hypertension, building a framework for future research towards improved prediction and prevention of premature development of CVD. We included 78 hypertensive and 79 normotensive participants from the African-PREDICT study (aged 20-30 years), matched for sex (51% male) and ethnicity (49% black and 51% white). Urinary peptidomics data were acquired using capillary-electrophoresis-time-of-flight-mass-spectrometry. Hypertension-associated peptides were identified and combined into a support vector machine-based multidimensional classifier. When comparing the peptide data between the normotensive and hypertensive groups, 129 peptides were nominally differentially abundant (Wilcoxon p < 0.05). Nonetheless, only three peptides, all derived from collagen alpha-1(III), remained significantly different after rigorous adjustments for multiple comparisons. The 37 most significant peptides (all p ≤ 0.001) served as basis for the development of a classifier, with 20 peptides being combined into a unifying score, resulting in an AUC of 0.85 in the ROC analysis (p < 0.001), with 83% sensitivity at 80% specificity. Our study suggests potential value of urinary peptides in the classification of hypertension, which could enable earlier diagnosis and better understanding of the pathophysiology of hypertension and premature cardiovascular disease development.

Urinary Peptidomics and Pulse Wave Velocity: The African-PREDICT Study.

Increased arterial stiffness is related to early vascular aging and is an independent predictor for cardiovascular disease and mortality. Molecular mechanisms underlying increased arterial stiffness are largely unexplored, especially at the proteome level. We aimed to explore the relationship between pulse wave velocity and urinary proteomics. We included 919 apparently healthy (no chronic illnesses) Black and White men and women (equally distributed) between 20 and 30 years from the African-PREDICT study. Capillary electrophoresis time-of-flight mass spectrometry was used to analyze the urinary proteome. We measured the carotid-femoral pulse wave velocity to estimate arterial stiffness. In the total group, pulse wave velocity correlated positively with collagen-derived peptides including collagen types I, II, III, IV, V, and IX and inversely with collagen type XI (adjusted for mean arterial pressure). Regarding noncollagen-derived peptides, pulse wave velocity positively correlated with polymeric immunoglobulin receptor peptides (n = 2) (all q-value ≤0.05). In multivariable adjusted analyses, pulse wave velocity associated positively and independently with seven urinary peptides (collagen type I, n = 5) (all p-value ≤0.05). We found significant positive and independent associations between pulse wave velocity and the collagen type I-derived peptides, suggesting that dysregulation of collagen type I in the extracellular matrix scaffold could lead to early onset of increased arterial stiffness.

Validation of diagnostic nomograms based on CE-MS urinary biomarkers to detect clinically significant prostate cancer.

PURPOSE: Prostate cancer (PCa) is one of the most common cancers and one of the leading causes of death worldwide. Thus, one major issue in PCa research is to accurately distinguish between indolent and clinically significant (csPCa) to reduce overdiagnosis and overtreatment. In this study, we aim to validate the usefulness of diagnostic nomograms (DN) to detect csPCa, based on previously published urinary biomarkers. METHODS: Capillary electrophoresis/mass spectrometry was employed to validate a previously published biomarker model based on 19 urinary peptides specific for csPCa. Added value of the 19-biomarker (BM) model was assessed in diagnostic nomograms including prostate-specific antigen (PSA), PSA density and the risk calculator from the European Randomized Study of Screening. For this purpose, urine samples from 147 PCa patients were collected prior to prostate biopsy and before performing digital rectal examination (DRE). The 19-BM score was estimated via a support vector machine-based software based on the pre-defined cutoff criterion of - 0.07. DNs were subsequently developed to assess added value of integrative diagnostics. RESULTS: Independent validation of the 19-BM resulted in an 87% sensitivity and 65% specificity, with an AUC of 0.81, outperforming PSA (AUC PSA: 0.64), PSA density (AUC PSAD: 0.64) and ERSPC-3/4 risk calculator (0.67). Integration of 19-BM with the rest clinical variables into distinct DN, resulted in improved (AUC range: 0.82-0.88) but not significantly better performances over 19-BM alone. CONCLUSION: 19-BM alone or upon integration with clinical variables into DN, might be useful for detecting csPCa by decreasing the number of biopsies.

CD99 and polymeric immunoglobulin receptor peptides deregulation in critical COVID-19: A potential link to molecular pathophysiology?

Identification of significant changes in urinary peptides may enable improved understanding of molecular disease mechanisms. We aimed towards identifying urinary peptides associated with critical course of COVID-19 to yield hypotheses on molecular pathophysiological mechanisms in disease development. In this multicentre prospective study urine samples of PCR-confirmed COVID-19 patients were collected in different centres across Europe. The urinary peptidome of 53 patients at WHO stages 6-8 and 66 at WHO stages 1-3 COVID-19 disease was analysed using capillary electrophoresis coupled to mass spectrometry. 593 peptides were identified significantly affected by disease severity. These peptides were compared with changes associated with kidney disease or heart failure. Similarities with kidney disease were observed, indicating comparable molecular mechanisms. In contrast, convincing similarity to heart failure could not be detected. The data for the first time showed deregulation of CD99 and polymeric immunoglobulin receptor peptides and of known peptides associated with kidney disease, including collagen and alpha-1-antitrypsin. Peptidomic findings were in line with the pathophysiology of COVID-19. The clinical corollary is that COVID-19 induces specific inflammation of numerous tissues including endothelial lining. Restoring these changes, especially in CD99, PIGR and alpha-1-antitripsin, may represent a valid and effective therapeutic approach in COVID-19, targeting improvement of endothelial integrity.

Preexisting hypertension and pregnancy-induced hypertension reveal molecular differences in placental proteome in rodents.

Preexisting or new onset of hypertension affects pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. In certain cases, it also leads to long-term maternal cardiovascular complications. The placenta is a key player in the pathogenesis of complicated hypertensive pregnancies, however the pathomechanisms leading to an abnormal placenta are poorly understood. In this study, we compared the placental proteome of two pregnant hypertensive models with their corresponding normotensive controls: a preexisting hypertension pregnancy model (stroke-prone spontaneously hypertensive rats; SHRSP) versus Wistar-Kyoto and the transgenic RAS activated gestational hypertension model (transgenic for human angiotensinogen Sprague-Dawley rats; SD-PE) versus Sprague-Dawley rats, respectively. Label-free proteomics using nano LC-MS/MS was performed for identification and quantification of proteins. Between the two models, we found widespread differences in the expression of placental proteins including those related to hypertension, inflammation, and trophoblast invasion, whereas pathways such as regulation of serine endopeptidase activity, tissue injury response, coagulation, and complement activation were enriched in both models. We present for the first time the placental proteome of SHRSP and SD-PE and provide insight into the molecular make-up of models of hypertensive pregnancy. Our study informs future research into specific preeclampsia and chronic hypertension pregnancy mechanisms and translation of rodent data to the clinic.

Proteome-based classification of Nonmuscle Invasive Bladder Cancer.

DNA/RNA-based classification of bladder cancer (BC) supports the existence of multiple molecular subtypes, while investigations at the protein level are scarce. Here, we aimed to investigate if Nonmuscle Invasive Bladder Cancer (NMIBC) can be stratified to biologically meaningful groups based on the proteome. Tissue specimens from 117 patients at primary diagnosis (98 with NMIBC and 19 with MIBC), were processed for high-resolution proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteomics output was subjected to unsupervised consensus clustering, principal component analysis (PCA) and investigation of subtype-specific features, pathways, and gene sets. NMIBC patients were optimally stratified to three NMIBC proteomic subtypes (NPS), differing in size, clinicopathologic and molecular backgrounds: NPS1 (mostly high stage/grade/risk samples) was the smallest in size (17/98) and overexpressed proteins reflective of an immune/inflammatory phenotype, involved in cell proliferation, unfolded protein response and DNA damage response, whereas NPS2 (mixed stage/grade/risk composition) presented with an infiltrated/mesenchymal profile. NPS3 was rich in luminal/differentiation markers, in line with its pathological composition (mostly low stage/grade/risk samples). PCA revealed a close proximity of NPS1 and conversely, remoteness of NPS3 to the proteome of MIBC. Proteins distinguishing these two extreme subtypes were also found to consistently differ at the mRNA levels between high and low-risk subtypes of the UROMOL and LUND cohorts. Collectively, our study identifies three proteomic NMIBC subtypes and following a cross-omics validation in two independent cohorts, shortlists molecular features meriting further investigation for their biomarker or potentially therapeutic value.

Bile and urine peptide marker profiles: access keys to molecular pathways and biological processes in cholangiocarcinoma.

BACKGROUND: Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. METHODS: Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. RESULTS: For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. CONCLUSIONS: The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. METHODS: LC-MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann-Whitney p value ≤ 0.05) and a minimum of twofold change. RESULTS: Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. CONCLUSIONS: This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD.

Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes.

Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene family of puc genes that encode the alpha and beta apoproteins, which form the LH2 complexes. A genetic dissection strategy has been adopted in order to try and understand which spectroscopic form of LH2 these different genes produce. This paper presents a characterisation of one of the deletion mutants generated in this program, the pucBAd only mutant. This mutant produces an unusual spectroscopic form of LH2 that only has a single large NIR absorption band at 800 nm. Spectroscopic and pigment analyses on this complex suggest that it has basically a similar overall structure as that of the wild-type HL LH2 complex. The mutant has the unique phenotype where the mutant LH2 complex is only produced when cells are grown at LL. At HL the mutant only produces the LH1-RC core complex.

Urinary proteomics for prediction of mortality in patients with type 2 diabetes and microalbuminuria.

BACKGROUND: The urinary proteomic classifier CKD273 has shown promise for prediction of progressive diabetic nephropathy (DN). Whether it is also a determinant of mortality and cardiovascular disease in patients with microalbuminuria (MA) is unknown. METHODS: Urine samples were obtained from 155 patients with type 2 diabetes and confirmed microalbuminuria. Proteomic analysis was undertaken using capillary electrophoresis coupled to mass spectrometry to determine the CKD273 classifier score. A previously defined CKD273 threshold of 0.343 for identification of DN was used to categorise the cohort in Kaplan-Meier and Cox regression models with all-cause mortality as the primary endpoint. Outcomes were traced through national health registers after 6 years. RESULTS: CKD273 correlated with urine albumin excretion rate (UAER) (r = 0.481, p = <0.001), age (r = 0.238, p = 0.003), coronary artery calcium (CAC) score (r = 0.236, p = 0.003), N-terminal pro-brain natriuretic peptide (NT-proBNP) (r = 0.190, p = 0.018) and estimated glomerular filtration rate (eGFR) (r = 0.265, p = 0.001). On multivariate analysis only UAER (ß = 0.402, p < 0.001) and eGFR (ß = - 0.184, p = 0.039) were statistically significant determinants of CKD273. Twenty participants died during follow-up. CKD273 was a determinant of mortality (log rank [Mantel-Cox] p = 0.004), and retained significance (p = 0.048) after adjustment for age, sex, blood pressure, NT-proBNP and CAC score in a Cox regression model. CONCLUSION: A multidimensional biomarker can provide information on outcomes associated with its primary diagnostic purpose. Here we demonstrate that the urinary proteomic classifier CKD273 is associated with mortality in individuals with type 2 diabetes and MA even when adjusted for other established cardiovascular and renal biomarkers.

Mitogen-Activated Protein Kinase 14 Promotes AKI.

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry-based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity-deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target.

Noninvasive diagnosis of chronic kidney diseases using urinary proteome analysis.

BACKGROUND: In spite of its invasive nature and risks, kidney biopsy is currently required for precise diagnosis of many chronic kidney diseases (CKDs). Here, we explored the hypothesis that analysis of the urinary proteome can discriminate different types of CKD irrespective of the underlying mechanism of disease. METHODS: We used data from the proteome analyses of 1180 urine samples from patients with different types of CKD, generated by capillary electrophoresis coupled to mass spectrometry. A set of 706 samples served as the discovery cohort, and 474 samples were used for independent validation. For each CKD type, peptide biomarkers were defined using statistical analysis adjusted for multiple testing. Potential biomarkers of statistical significance were combined in support vector machine (SVM)-based classifiers. RESULTS: For seven different types of CKD, several potential urinary biomarker peptides (ranging from 116 to 619 peptides) were defined and combined into SVM-based classifiers specific for each CKD. These classifiers were validated in an independent cohort and showed good to excellent accuracy for discrimination of one CKD type from the others (area under the receiver operating characteristic curve ranged from 0.77 to 0.95). Sequence analysis of the biomarkers provided further information that may clarify the underlying pathophysiology. CONCLUSIONS: Our data indicate that urinary proteome analysis has the potential to identify various types of CKD defined by pathological assessment of renal biopsies and current clinical practice in general. Moreover, these approaches may provide information to model molecular changes per CKD.

A urinary proteome-based classifier for the early detection of decline in glomerular filtration.

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 <29 mL/min/1.73 m 2 . In addition, areas under the curve for CKD273 and UAE were calculated. RESULTS: In early stage CKD, the urinary proteome-based classifier performed significantly better than UAE in detecting progressors. In contrast, UAE performed better in patients with late-stage CKD. No significant difference in performance was found between CKD273 and UAE in patients with moderately reduced renal function. CONCLUSIONS: These results suggest that urinary peptides, as combined in the CKD273 classifier, allow the detection of progressive CKD at early stages, a point where therapeutic intervention is more likely to be effective. However, late-stage disease, where irreversible damage of the kidney is already present, is better detected by UAE.

High-Throughput LC-MS/MS Proteomic Analysis of a Mouse Model of Mesiotemporal Lobe Epilepsy Predicts Microglial Activation Underlying Disease Development.

Uncovering the molecular mechanisms of mesiotemporal lobe epilepsy (MTLE) is critical to identify therapeutic targets. In this study, we performed global protein expression analysis of a kainic acid (KA) MTLE mouse model at various time-points (1, 3, and 30 days post-KA injection -dpi), representing specific stages of the syndrome. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination with label-free protein quantification using three processing approaches for quantification, was applied. Following comparison of KA versus NaCl-injected mice, 22, 53, and 175 proteins were differentially (statistically significant) expressed at 1, 3 and 30dpi, respectively, according to all three quantification approaches. Selected findings were confirmed by multiple reaction monitoring LC-MS/MS. As a positive control, the astrocyte marker GFAP was found to be upregulated (3dpi: 1.9 fold; 30dpi: 12.5 fold), also verified by IHC. The results collectively suggest that impairment in synaptic transmission occurs even right after initial status epilepticus (1dpi), with neurodegeneration becoming more extensive during epileptogenesis (3dpi) and sustained at the chronic phase (30dpi), where also extensive glial- and astrocyte-mediated inflammation is evident. This molecular profile is in line with observed phenotypic changes in human MTLE, providing the basis for future studies on new molecular targets for the disease.

Proteomics of vitreous in neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (nAMD) has been described as a predominantly inflammatory and proangiogenic retino-choroidal disease. Vitreous humor (VH) is the adjacent and accessible compartment which, due to the vicinity to the retina, might best represent changes of protein-based mediators of nAMD. The aim of this clinical-experimental study was to analyze the nAMD associated VH proteome of previously untreated patients whilst taking different groups of nAMD into account, based on their clinical presentation (clinical diagnosis groups). Electrophoresis coupled online to mass spectrometry (CE-MS) as well as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were used to analyze VH of 108 nAMD patients and 24 controls with idiopathic floaters. A total of 101 different proteins with at least two unique peptides could be identified. Using a stringent statistical analysis with implementation of the closed test principle, we were able to identify four proteins that may be involved in the pathophysiology of nAMD: Clusterin, opticin, pigment epithelium-derived factor and prostaglandin-H2 d-isomerase. Using independent samples, ROC-Area under the curve was determined proving the validity of the results: Clusterin 0.747, opticin 0.656, pigment epithelium-derived factor 0.514, prostaglandin-H2 d-isomerase 0.712. In addition, validation through ELISA measurements was performed. The identified proteins may serve as potential biomarkers or even targets of therapy for nAMD.

Acute kidney injury prediction in cardiac surgery patients by a urinary peptide pattern: a case-control validation study.

BACKGROUND: Acute kidney injury (AKI) is a prominent problem in hospitalized patients and associated with increased morbidity and mortality. Clinical medicine is currently hampered by the lack of accurate and early biomarkers for diagnosis of AKI and the evaluation of the severity of the disease. In 2010, we established a multivariate peptide marker pattern consisting of 20 naturally occurring urinary peptides to screen patients for early signs of renal failure. The current study now aims to evaluate if, in a different study population and potentially various AKI causes, AKI can be detected early and accurately by proteome analysis. METHODS: Urine samples from 60 patients who developed AKI after cardiac surgery were analyzed by capillary electrophoresis-mass spectrometry (CE-MS). The obtained peptide profiles were screened by the AKI peptide marker panel for early signs of AKI. Accuracy of the proteomic model in this patient collective was compared to that based on urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) ELISA levels. Sixty patients who did not develop AKI served as negative controls. RESULTS: From the 120 patients, 110 were successfully analyzed by CE-MS (59 with AKI, 51 controls). Application of the AKI panel demonstrated an AUC in receiver operating characteristics (ROC) analysis of 0.81 (95 % confidence interval: 0.72-0.88). Compared to the proteomic model, ROC analysis revealed poorer classification accuracy of NGAL and KIM-1 with the respective AUC values being outside the statistical significant range (0.63 for NGAL and 0.57 for KIM-1). CONCLUSIONS: This study gives further proof for the general applicability of our proteomic multimarker model for early and accurate prediction of AKI irrespective of its underlying disease cause.

Diagnosis and Prediction of CKD Progression by Assessment of Urinary Peptides.

Progressive CKD is generally detected at a late stage by a sustained decline in eGFR and/or the presence of significant albuminuria. With the aim of early and improved risk stratification of patients with CKD, we studied urinary peptides in a large cross-sectional multicenter cohort of 1990 individuals, including 522 with follow-up data, using proteome analysis. We validated that a previously established multipeptide urinary biomarker classifier performed significantly better in detecting and predicting progression of CKD than the current clinical standard, urinary albumin. The classifier was also more sensitive for identifying patients with rapidly progressing CKD. Compared with the combination of baseline eGFR and albuminuria (area under the curve [AUC]=0.758), the addition of the multipeptide biomarker classifier significantly improved CKD risk prediction (AUC=0.831) as assessed by the net reclassification index (0.303±-0.065; P<0.001) and integrated discrimination improvement (0.058±0.014; P<0.001). Correlation of individual urinary peptides with CKD stage and progression showed that the peptides that associated with CKD, irrespective of CKD stage or CKD progression, were either fragments of the major circulating proteins, suggesting failure of the glomerular filtration barrier sieving properties, or different collagen fragments, suggesting accumulation of intrarenal extracellular matrix. Furthermore, protein fragments associated with progression of CKD originated mostly from proteins related to inflammation and tissue repair. Results of this study suggest that urinary proteome analysis might significantly improve the current state of the art of CKD detection and outcome prediction and that identification of the urinary peptides allows insight into various ongoing pathophysiologic processes in CKD.

Tomato Juice Consumption Modifies the Urinary Peptide Profile in Sprague-Dawley Rats with Induced Hepatic Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in Western countries, with a high prevalence, and has been shown to increase the risk of type 2 diabetes, cardiovascular disease (CVD), etc. Tomato products contain several natural antioxidants, including lycopene-which has displayed a preventive effect on the development of steatosis and CVD. Accordingly, the aim of the present work was to evaluate the effect of tomato juice consumption on the urinary peptide profile in rats with NAFLD induced by an atherogenic diet and to identify potential peptide biomarkers for diagnosis. Urine samples, collected weekly for four weeks, were analyzed by capillary electrophoresis (CE) coupled to a mass spectrometer (MS). A partial least squares-discriminant analysis (PLS-DA) was carried out to explore the association between differential peptides and treatments. Among the 888 peptides initially identified, a total of 55 were obtained as potential biomarkers. Rats with steatosis after tomato juice intake showed a profile intermediate between that of healthy rats and that of rats with induced hepatic steatosis. Accordingly, tomato products could be considered as a dietary strategy for the impairment of NAFLD, although further research should be carried out to develop a specific biomarkers panel for NAFLD.

Cerebrospinal fluid prohormone processing and neuropeptides stimulating feed intake of dairy cows during early lactation.

After parturition, feed intake of dairy cows increases within the first weeks of lactation, but the molecular mechanisms stimulating or delaying the slope of increase are poorly understood. Some of the molecules controlling feed intake are neuropeptides that are synthesized as propeptides and subsequently processed before they bind to specific receptors in feeding centers of the brain. Cerebrospinal fluid surrounds most of the feed intake regulatory centers and contains numerous neuropeptides. In the present study, we used a proteomic approach to analyze the neuropeptide concentrations in cerebrospinal fluid taken from dairy cows between day -18 and -10, and between day +10 and +20 relative to parturition. We found 13 proteins which were only present in samples taken before parturition, 13 proteins which were only present in samples taken after parturition, and 25 proteins which were commonly present, before and after parturition. Among them, differences in pro-neuropeptide Y, proenkephalin-A, neuroendocrine convertase-2, neurosecretory protein VGF, chromogranin-A, and secretogranin-1 and -3 concentrations relative to parturition highlight propeptides and prohormone processings involved in the control of feed intake and energy homeostasis. Scaffold analysis further emphasized an increased tone of endogenous opioids associated with the postparturient increase of feed intake.