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Resumen: Antecedentes y objetivo: Las vesículas extracelulares (VEs) reflejan el estado fisiopatológico de sus células de origen y constituyen un reservorio de información renal accesible en la orina. Cuando la biopsia no es una opción, las VEs se presentan como un centinela de funcionalidad y daño, constituyendo una aproximación no invasiva. Sin embargo, el análisis de las VEs de la orina requiere de un aislamiento previo que ralentiza y dificulta su traslación a la práctica clínica. El objetivo de este trabajo es mostrar la aplicabilidad de la tecnología «sensor de imágenes de reflectancia interferométrica de una sola partícula» (SP-IRIS) mediante la plataforma ExoView® para el análisis directo de las VEs de la orina y de proteínas implicadas en funcionalidad renal. Materiales y métodos: La tecnología ExoView® permite la cuantificación y el fenotipado de las VEs presentes en la orina y la cuantificación de sus proteínas, de membrana e internas. En este trabajo se aplica esta tecnología a la cuantificación de las VEs de la orina y de sus proteínas con expresión renal tubular, amnionless (AMN) y secreted frizzled-related protein 1 (SFRP1), empleando únicamente 5 μl de orina. La expresión tubular se confirmó por inmunohistoquímica. Resultados: El tamaño medio de las VEs analizadas fue de 59 ± 16 nm para las capturadas por la tetraspanina CD63, 61 ± 16 nm para las capturadas por la tetraspanina CD81 y 59 ± 10 nm para la tetraspanina CD9, siendo CD63 la subpoblación de VEs mayoritaria en la orina (48,92%). La distribución de AMN y SFRP1 en las 3 tetraspaninas de captura resultó ser similar para ambas proteínas, expresándose mayoritariamente en CD63 (48,23% para AMN y 52,1% para SFRP1). Conclusiones: Este trabajo evidencia la aplicabilidad y las ventajas de la técnica ExoView® para el análisis directo de las VEs de la orina, y su cargo proteico en relación con el túbulo renal. El empleo de volúmenes mínimos, 5 μl, y el tiempo total de análisis no superior a 3 h facilita la traslación a la práctica clínica diaria de las VEs como fuente de información diagnóstica. Abstract: Background and objective: Extracellular vesicles (EVs) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EVs present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EVs in urine requires prior isolation, which slows down and hinders translation to clinical practice. The aim of this study is to show the applicability of the “single particle interferometric reflectance imaging sensor” (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EVs and proteins involved in renal function. Materials and methods: The ExoView® technology enables the quantification and phenotyping of EVs present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EVs and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5 μl of urine. Tubular expression was confirmed by immunohistochemistry. Results: The mean size of the EVs analysed was 59 ± 16 nm for those captured by tetraspanin CD63, 61 ± 16 nm for those captured by tetraspanin CD81, and 59 ± 10 nm for tetraspanin CD9, with CD63 being the majority EVs subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1). Conclusions: This work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EVs and their protein content in relation to the renal tubule. The use of minimum volumes, 5 μl, and the total analysis time not exceeding three hours facilitate the translation of EVs to daily clinical practice as source of diagnostic information.

Abstract Background Current definition of chronic kidney disease (CKD) identifies only advanced stages, but effective management demands early detection. Urinary albumin‐to‐creatinine ratio (ACR) 30 mg/g is a cut‐off point for CKD clinical diagnosis. Patients with lower values (normoalbuminuria) and eGFR > 60 mL/min/1.73 m2 are considered at no increased cardiorenal risk. However, higher incidence of renal function decline and cardiovascular events have been shown within the normoalbuminuria range. Novel subclinical indicators may help to identify higher‐risk patients. Urinary extracellular vesicles (uEVs) are sentinels of renal function non‐invasively. Here we aimed to approach the early assessment of cardiorenal risk by investigating the protein cargo of uEVs. Methods Hypertensive patients were classified in control group (C) with ACR 6200 uEVs proteins identified, 43 define a panel significantly altered in HN patients without variation in urine, mostly annotated in the tubule (39 out of 43). The tubular transporter long‐chain fatty acid transport protein 2 (SLC27A2) and the apical membrane protein amnionless (AMN) confirmed their alteration in HN patients evidencing impaired tubular reabsorption. SLC27A2 showed tubular expression and significantly reduced levels in patients with diagnostic criteria for CKD. Conclusions Alterations in the EV‐mediated molecular profile are evident before pathological ACR levels are reached. Direct quantitation of SLC27A2 and AMN in uEVs helps identifying normoalbuminuric subjects with higher cardiorenal risk in early monitoring of CKD.

Moderately increased albuminuria, defined by an albumin to creatinine ratio (ACR) > 30 mg/g, is an indicator of subclinical organ damage associated with a higher risk of cardiovascular and renal disease. Normoalbuminuric subjects are considered at no cardiorenal risk in clinical practice, and molecular changes underlying early development are unclear. To decipher subjacent mechanisms, we stratified the normoalbuminuria condition. A total of 37 hypertensive patients under chronic renin–angiotensin system (RAS) suppression with ACR values in the normoalbuminuria range were included and classified as control (C) (ACR < 10 mg/g) and high-normal (HN) (ACR = 10–30 mg/g). Target metabolomic analysis was carried out by liquid chromatography and mass spectrometry to investigate the role of the cardiorenal risk urinary metabolites previously identified. Besides this, urinary free fatty acids (FFAs), fatty acid binding protein 1 (FABP1) and nephrin were analyzed by colorimetric and ELISA assays. A Mann–Whitney test was applied, ROC curves were calculated and Spearman correlation analysis was carried out. Nine metabolites showed significantly altered abundance in HN versus C, and urinary FFAs and FABP1 increased in HN group, pointing to dysregulation in the tricarboxylic acid cycle (TCA) cycle and fatty acids β-oxidation. We showed here how cardiorenal metabolites associate with albuminuria, already in the normoalbuminuric range, evidencing early renal damage at a tubular level and suggesting increased β-oxidation to potentially counteract fatty acids overload in the HN range.

Acute Kidney Injury (AKI) affects up to 30% of the patients who undergo cardiac surgery (CVS) and is related to higher mortality. We aim to investigate molecular features associated with in-hospital AKI development and determine the predictive value of these features when analyzed preoperatively. This is a case-control study. From an initial cohort of 110 recruited subjects, a total of 60 patients undergoing cardiac surgery were included: 20 (33%) developed in-hospital AKI (CVS-AKI) and 40 did not (controls, CVS-C). Pre- and post-surgery samples were collected and a prospective study was carried out. A total of 312 serum samples and 258 urine samples were analyzed by nuclear magnetic resonance, mass spectrometry and ELISA. Six features predicted AKI development in pre-surgery samples: urinary kidney functional loss marker kidney injury molecule-1 (uKIM-1), 2-hydroxybutyric acid, 2-hydroxyphenylacetic acid, hippuric acid, phosphoethanolamine and spermidine. Two of them stood out as powerful predictors. Pre-surgery uKIM-1 levels were increased in CVS-AKI vs. CVS-C (AUC = 0.721, p-value = 0.0392) and associated strongly with the outcome (OR = 5.333, p-value = 0.0264). Spermidine showed higher concentration in CVS-AKI (p-value < 0.0001, AUC = 0.970) and had a strong association with the outcome (OR = 69.75, p-value < 0.0001). uKIM-1 and particularly spermidine predict in-hospital AKI associated with CVS in preoperative samples. These findings may aid in preventing postoperative AKI and improve prognosis of CVS.

Pancreatogenic diabetes mellitus (T3cDM) is a highly frequent complication of pancreatic disease, especially chronic pancreatitis, and it is often misdiagnosed as type 2 diabetes mellitus (T2DM). A correct diagnosis allows the appropriate treatment of these patients, improving their quality of life, and various technologies have been employed over recent years to search for specific biomarkers of each disease. The main aim of this metabolomic project was to find differential metabolites between T3cDM and T2DM. Reverse-phase liquid chromatography coupled to high-resolution mass spectrometry was performed in serum samples from patients with T3cDM and T2DM. Multivariate Principal Component and Partial Least Squares-Discriminant analyses were employed to evaluate between-group variations. Univariate and multivariate analyses were used to identify potential candidates and the area under the receiver-operating characteristic (ROC) curve was calculated to evaluate their diagnostic value. A panel of five differential metabolites obtained an area under the ROC curve of 0.946. In this study, we demonstrate the usefulness of untargeted metabolomics for the differential diagnosis between T3cDM and T2DM and propose a panel of five metabolites that appear altered in the comparison between patients with these diseases.

Neoadjuvant chemotherapy (NACT) outcomes vary according to breast cancer (BC) subtype. Since pathologic complete response is one of the most important target endpoints of NACT, further investigation of NACT outcomes in BC is crucial. Thus, identifying sensitive and specific predictors of treatment response for each phenotype would enable early detection of chemoresistance and residual disease, decreasing exposures to ineffective therapies and enhancing overall survival rates. We used liquid chromatography−high‐resolution mass spectrometry (LC‐HRMS)‐based untargeted metabolomics to detect molecular changes in plasma of three different BC subtypes following the same NACT regimen, with the aim of searching for potential predictors of response. The metabolomics data set was analyzed by combining univariate and multivariate statistical strategies. By using ANOVA–simultaneous component analysis (ASCA), we were able to determine the prognostic value of potential biomarker candidates of response to NACT in the triple‐negative (TN) subtype. Higher concentrations of docosahexaenoic acid and secondary bile acids were found at basal and presurgery samples, respectively, in the responders group. In addition, the glycohyocholic and glycodeoxycholic acids were able to classify TN patients according to response to treatment and overall survival with an area under the curve model > 0.77. In relation to luminal B (LB) and HER2+ subjects, it should be noted that significant differences were related to time and individual factors. Specifically, tryptophan was identified to be decreased over time in HER2+ patients, whereas LysoPE (22:6) appeared to be increased, but could not be associated with response to NACT. Therefore, the combination of untargeted‐based metabolomics along with longitudinal statistical approaches may represent a very useful tool for the improvement of treatment and in administering a more personalized BC follow‐up in the clinical practice.