Urinary exosomal miRNAs in hypertension: methodological advancements and potential applications for biomarker discovery

Introduzione: Gli esosomi sono vescicole contenenti citoplasma rilasciate da molti tipi cellulari e che possono essere rinvenute in molti fluidi biologici, urine incluse. Gli esosomi urinari derivano dalle cellule epiteliali del tubulo renale e possono essere rilasciati da ogni segmento del nefrone. Gli esosomi urinari contengono constitutivamente RNA (piccoli RNA, microRNA ed mRNA) e possiedono un subset peculiare di proteine. I microRNA (microRNAs) sono piccole (20-22 nucleotidi) molecole di RNA endogeno non codificante che modulano l\u2019espressione genica. I miRNA modulano meccanismi regolatori che influenzano processi cellulari fondamentali come la differenziazione, la proliferazione, la morte e il metabolismo cellulare e la fisiopatologia di molte malattie. Il potenziale dei miRNA come biomarcatori diagnostici \ue8 stato recentemente dimostrato in una grande variet\ue0 di patologie. Lo scopo della presente ricerca si \ue8 focalizzato in modo selettivo sul possibile ruolo dei miRNA nell\u2019ipertensione umana, in particolare su una specifica forma di ipertensione secondaria, l\u2019aldosteronismo primario (PA). PA \ue8 la causa principale di ipertensione arteriosa secondaria, con una prevalenza stimata che varia dal 10 al 15% nella popolazione afferente a centri di ipertensione specialistici. Una diagnosi corretta di PA richiede il campionamento venoso surrenalico (AVS) per la classificazione del sottotipo (adenoma secernente aldosterone, APA oppure iperplasia surrenalica bilaterale, BAH). Dal momento che questo tipo di test non \ue8 facilmente praticabile, \ue8 invasivo e richiede capacit\ue0 specifiche dei radiologi interventisti, l\u2019identificazione di biomarcatori appropriati e specialmente non invasivi per la definizione del sottotipo \ue8 altamente auspicabile. Questa necessit\ue0 pratica \ue8 direttamente proporzionale alla crescente riscontro di pazienti affetti da PA. Per i suddetti motivi, la nostra ipotesi di lavoro \ue8 stata quella di identificare miRNA aventi una valenza diagnostica e/o prognostica negli esosomi urinari di pazienti con PA . Gli obiettivi di questo studio erano molti e possono essere riassunti come segue: i) definire il protocollo ottimale per isolare, purificare e validare gli esosomi dalle urine, ii) delineare il protocollo ottimale per una analisi su larga scala dei miRNA esosomiali, iii) analizzare i profili di espressione in sottogruppi di pazienti PA (APA e BAH) e con ipertensione essenziale (EH), e iv) studiare negli esosomi urinari l\u2019abbondanza di alcune proteine note per essere associate ad un anomalo trasporto del sodio a livello del tubulo renale in corso di malattia.. Metodi: Gli esosomi urinari sono stati isolati usando tecniche di ultrafiltrazione, ultracentrifugazione ed il reagente ExoQuick-TC\u2122. La caratterizzazione delle vescicole purificate \ue8 stata ottenuta attraverso analisi con \u201cdynamic light scattering\u201d per la determinazione della dimensione e con Western Immunoblotting per la determinazione del marcatore esosomiale Acquaporina 2 (AQP2). Allo scopo di ottimizzare il protocollo di purificazione dei miRNA dagli esosomi urinari, abbiamo paragonato i tre diversi metodi per l\u2019isolamento degli esosomi urinari e le tecniche di estrazione dell\u2019RNA. La resa, la dimensione e la qualit\ue0 dell\u2019 RNA esosomiale \ue8 stata valutata rispettivamente mediante colorante fluorescente, elettroforesi capillare e parametri spettrofotometrici. La valutazione della presenza e dell\u2019abbondanza dei miRNA \ue8 stata ottenuta attraverso analisi con RT-qPCR. Lo studio dei profili di espressione dei miRNA da esosomi urinari \ue8 stato eseguito su un totale di 12 campioni (gruppo test: 4APA, 4BAH e 4 EH). I profili di espressione ottenuti con cartucce microfluidiche \u201cTaqMan\u2122 human Low density array (A&B)\u201d sono stati valutati allo scopo di identificare diversi profili di miRNA. I miRNA con livelli di espressione significativamente diversi tra i vari gruppi sperimentali (APA,BAH ed EH) sono stati validati attraverso RT-qPCR. Gli array TaqMan\u2122 hanno mostrato alcuni miRNA che risultavano espressi a livelli costanti, e che quindi potevano essere utilizzati come controlli endogeni per gli esosomi urinari. La predizione dei target dei miRNA \ue8 stata effettuata utilizzando diversi algoritmi computazionali (Target Scan, Pictar e miRandola). E\u2019 stata inoltre valutata con Western Immunoblotting l\u2019abbondanza relativa delle proteine NCC (cotrasportatore di cloruro di sodio) e prostasina, in pazienti affetti da PA. Risultati: Abbiamo confermato la natura esosomiale di tutte le frazioni di nanovescicole isolate utilizzando tutti e tre i metodi (Ultrafiltrazione, Ultracentrifugazione ed ExoQuick-TC\u2122). L\u2019ultrafiltrazione, d\u2019altra parte, \ue8 risultata essere il metodo pi\uf9 consono per l\u2019isolamento di esosomi urinari. La resa pi\uf9 alta di RNA esosomiale quantificata con il metodo di colorazione RiboGreen\uae \ue8 stata ottenuta utilizzando una combinazione dei metodi TRI Reagent\u2122 e miRNeasy\uae, seguita da un secondo passaggio rispettivamente con TRI Reagent\u2122, SeraMir\u2122, miRCURY\u2122, mirVana\u2122 e miRNeasy\uae, e dopo analisi multivariata, il metodo SeraMir\u2122 \ue8 risultato essere il migliore all\u2019analisi quantitativa tramite RT-qPCR in termini di resa in miRNA, purezza ed accuratezza. E\u2019 stata inoltre analizzata l\u2019influenza delle condizioni di stoccaggio del campione, arrivando alla conclusione che l\u2019abbondanza relativa in termini di miRNA non \ue8 penalizzata dalla conservazione. L\u2019analisi di \u201cprofiling\u201d dei miRNA attraverso TaqMan\u2122 arrays ha portato al riscontro di 132 miRNA differenzialmente espressi negli esosomi urinari tra i due gruppi di ipertesi, PA (APA e BAH) ed EH. Dopo la successiva validazione, il miR-139-3p ed il miR-499-3p in APA, il miR-1179 ed il miR-141# in BAH sono stati identificati come potenziali biomarcatori. Il miRNA let-7c, invece, \ue8 stato riscontrato essere espresso in modo costante sia in tutti i gruppi analizzati sia nei controlli, suggerendo un suo potenziale ruolo come controllo endogeno.. Nei pazienti APA, il contenuto esosomiale di prostasina \ue8 stato trovato essere significativamente pi\uf9 alto rispetto ai campioni dei soggetti EH (P<0.05); inoltre si \ue8 osservata una drastica riduzione della quantit\ue0 di prostasina esosomiale dopo intervento di surrenalectomia. Un elevato carico salino (nel contesto del test di conferma per la diagnosi di PA) si \ue8 rivelato influire sull\u2019abbondanza di proteina NCC, causandone una significativa riduzione Conclusioni: La metodica di ultrafiltrazione combinata con l\u2019uso delle colonne SeraMir\u2122 exoRNA si \ue8 dimostrata essere la procedura ottimale per una rapida, economica ed efficiente purificazione dei miRNAs dagli esosomi urinari, utilizzabile per future applicazioni di ricerca nel campo dei miRNAsInoltre, le differenze nel profilo di espressione dei miRNAs fra le diverse patologie studiate, APA, BAH e EH, e il riscontro di miRNAs specifici per patologia (miR-139-3p e miR-499-3p in APA; miR-1179 and miR-141# in BAH) possono rappresentare dei potenziali biomarcatori in grado di permettere l\u2019identificazione e la differenziazione di pazienti affetti da PA ed EH. Due proteine sono state identificate negli esosomi urinari, la prostasina, una proteasi associata all\u2019attivazione del canale epiteliale del sodio (ENaC), e l\u2019 NCC, ed entrambe rappresentano marcatori promettenti per futuri studi riguardanti il PA. Questo studio infine conferma il potenziale dell\u2019analisi del profilo dei miRNAs come innovativo strumento diagnostico nel contesto dell\u2019ipertensione secondaria, in grado di permettere l\u2019identificazione dei sottogruppi di PA. Questa analisi non invasiva, basata sui miRNAs degli esosomi urinari, permetterebbe anche una migliore partecipazione dei pazienti allo screening, paragonata ad altre tecniche invasive (CT e AVS), sebbene l\u2019analisi di gruppi pi\uf9 numerosi di soggetti sia necessaria per la conferma dei nostri risultati.Background: Exosomes are cytoplasm containing vesicles released by many cell types that can be found in several biological fluids including urine. Urinary exosomes are derived from renal tubular epithelial cells and can be released from every segment of the nephron. Urinary exosomes constitutively contain RNA (small RNAs, microRNAs and mRNAs) and harbour unique subset of proteins. MicroRNAs (miRNAs) are endogenous short (20\u201322 nucleotides) non-coding RNA molecules that mediate gene expression. miRNAs modulated regulatory mechanisms influence fundamental cellular processes such as differentiation, proliferation, death, metabolism and pathophysiology of many diseases. The potential of miRNAs as diagnostic biomarkers has recently been shown in a broad variety of diseases. The focus of the present research was selectively centred on the possible role of miRNAs in human hypertension, particularly in a specific secondary form of hypertension i.e. Primary Aldosteronism (PA). PA is the major cause of secondary arterial hypertension with an estimated prevalence ranging from 10 to 15% in populations referred to specialist hypertension units. A correct diagnosis of PA requires adrenal venous sampling (AVS) for the classification of subtype (aldosterone producing adenoma, APA or bilateral adrenal hyperplasia, BAH). Since such testing is not easily suitable, is invasive and requires specialist skill of interventional radiologists; appropriate and especially non-invasive biomarkers for the definition of subtype are highly desirable. Such practical need is directly proportional to the growing frequency of patients recognized to be affected by PA. For all these reasons, our working hypothesis was to identify miRNAs in urinary exosomes of PA patients with diagnostic and/or pathogenetic value; in the present thesis are reported the results so far obtained. The objectives of the study were multiple, and in a progressive logical order may be summarized as follows: i) to establish the optimal protocol to isolate, purify and validate exosomes from urine, ii) to establish the optimal protocol for high throughput analysis of exosomal miRNAs, iii) to perform miRNA expression profiling in subgroups of PA patients (APA and BAH) and essential hypertension (EH), and iv) to analyze urinary exosomal abundance of some proteins previously reported to be associated with an abnormal sodium handling at renal tubular level in course of PA. Methods: Urinary exosomes were isolated using Ultrafiltration, Ultracentrifugation and ExoQuick-TC\u2122 reagent. Characterization of the purified vesicles was done by dynamic light scattering analysis (DLS) for size determination and Western Immunoblotting to detect exosomal marker, aquaporin 2 (AQP2). To optimize the urinary exosomal miRNA purification protocol, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR. Urinary exosomal miRNA profiling studies included a total of 12 test cohort samples (4 APA, 4 BAH and 4 EH). miRNA expression profiling using TaqMan\u2122 human Low density array microfluidic cards (A&B) was performed to identify differential miRNA profiles. miRNAs with significant differences in expression between experimental groups (APA, BAH and EH) were validated by real-time quantitative reverse-transcription PCR. Validation cohorts included both hypertensive and healthy subjects (n=10). TaqMan\u2122 arrays also identified some miRNAs that were expressed at constant levels, and hence can be used as urinary exosomal endogenous controls. miRNAs target prediction was carried out by Target Scan, Pictar and miRandola computational algorithms. We also examined urinary exosomal sodium chloride co-transporter (NCC) and Prostasin in patients with PA by western immunoblotting. Results: Urinary nanovesicles isolated using all the three methods (Ultrafiltration, Ultracentrifugation and ExoQuick-TC\u2122), confirmed the purified fractions as exosomes. However, Ultrafiltration resulted to be the most suited method for urinary exosome isolation. The highest exosomal RNA yield quantified by RiboGreen\uae staining was obtained with the combination of TRI Reagent\u2122 with miRNeasy\uae, followed by TRI Reagent\u2122, SeraMir\u2122, miRCURY\u2122, mirVana\u2122 and miRNeasy\uae; but after a multivariate analysis, SeraMir\u2122 scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. TaqMan\u2122 arrays miRNA profiling analysis revealed a total of 132 differentially expressed urinary exosomal miRNAs between PA (APA and BAH) and EH groups. On further validation, miR-139-3p and miR-499-3p in APA, miR-1179 and miR-141# in BAH were identified as potential biomarkers. Let-7c was constantly expressed in all tested groups and controls, suggesting its potential role as urinary exosomal endogenous control for miRNA studies. In APA patients, urinary exosomal content of Prostasin was significantly higher (P<0.05) than in EH patients and it drastically lowered after adrenalectomy. A high sodium load (in context of saline loading test) resulted in significant decrease in urinary exosomal NCC. Conclusions: Ultrafiltration in combination with SeraMir\u2122 exoRNA columns resulted to be the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes which can be perfectly suited for further applicative research in the field of miRNAs research. Urinary exosome samples can be used for the analysis of miRNA expression in PA. Though, miRNA expression profiles differ between APA, BAH and EH groups, pathology specific miRNAs (miR-139-3p and miR-499-3p in APA; miR-1179 and miR-141# in BAH) may serve as potential biomarkers for identifying and differentiating subgroups of PA and EH patients. Urinary exosomal Prostasin, a protease associated with ENaC activation, and NCC are promising markers for future PA research. This study also confirms the power of miRNA profiling as a novel diagnostic tool for the diagnosis of secondary forms of hypertension and can assist in separating subgroups of PA. Such non-invasive urinary exosomal-based miRNA assays could provide better screening compliance as compared to invasive screening methods (CT and AVS) but larger cohorts are needed to further confirm our preliminary results

Isolation of Urinary Exosomal miRNAs: Comparative analysis of different methods

Exosomes can be detected in urine and are released from every segment of the nephron. Urinary exosomes harbor unique subset of proteins, reflecting their cellular source, and constitutively contain RNA. The presence of large amounts of small RNAs in exosomes suggests that exosomes may contain specific microRNAs that provide valuable information as noninvasive clinical biomarkers in both diagnostic and prognostic areas for patients with renal pathologies [1]. We report a comparative analysis of different methods for the isolation of urinary exosomes and the analysis of their microRNA content. We applied and compared several methodologies, including nanomembrane concentrators and Exoquick-TCTM precipitation reagent [2] for exosome purification; TRI reagent and various commercial kits (Qiagen, Ambion, miRcury and Seramir) for exosomal RNAs extraction. Purified RNA was subjected to standard validation such as nanodrop quantification, ribo-pico green measurements and bioanalyser analysis. Moreover all the samples were checked for microRNAs/mRNA/small RNAs content by RT-qPCR specific assays. Based on these results we selected the most reliable and convenient method for microRNAs extraction from urinary exosomes. The selection of the appropriate exosomal miRNA isolation method was dependent on the validation results in terms of RNA yield and the absence of contaminants

Urinary Exosomal miRNAs: Best Strategies for Isolation and Analysis

Background & Aims: Urinary exosomes are released from every segment of the nephron and harbor unique subset of proteins and RNA. The abundance of small RNAs in exosomes provides a beneficial \u201ctool\u201d to explore specific microRNAs which, in turn could be helpful as noninvasive clinical biomarkers in cardiovascular pathologies [1]. MicroRNAs are endogenous, small (20-22 nucleotides), non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs [2]. The aim of this study was to compare in detail the available strategies for isolation of urinary exosomal microRNAs in combination with different methods for RNA purification. Methods: Urinary Exosomes were isolated using Ultrafiltration (Nanomembrane Concentrators), and Exoquick-TCTM precipitation reagent [3]. Exosomal RNA was isolated using TRI reagent and various commercial kits (Qiagen, Ambion, miRcury and Seramir). Purified total RNA was quantified using Ribogreen, small RNA quantification was obtained using Agilent Bioanalyzer and target miRNAs/mRNAs validation was done by RT-qPCR specific assays. Results: The combination of ultrafiltration for exosomes isolation; and Trizol-miRNeasy for exosomal RNA proved to be efficient compared with all the other methods. Conclusions: The selection of the appropriate urinary exosomal microRNAs isolation method was dependent on the total RNA yield, RNA purity and microRNA abundance. Further analysis in larger groups is required to reconfirm the efficiency of the developed method

Isolation of Urinary Exosomal miRNAs using various methods

Exosomes are involved in a wide spectrum of physiological mechanisms such as immune system modulation, paracrine functions and cell to cell communications. They can be detected in urine being released from every segment of the nephron. Urinary exosomes (UEs) constitutively contain RNA (microRNAs/mRNA/small RNAs) and harbour unique subset of proteins, reflecting their cellular source. With the aim to establish the best method both for urinary exosomes isolation and for the subsequent RNA profiling analysis, we compared three different UEs isolation methods and six RNA extraction techniques. Exosomal RNA yield, quality and size were assessed respectively by specific staining with fluorescent dyes (Ribogreen\uae, RNA specific quantification kit), spectrophotometric quantification (Nanodrop\uae ND \u2013 1000 spectrophotometer) and capillary electrophoresis (Agilent 2100 Bioanalyzer). All the samples were analysed for detection of selected miRNAs and mRNAs by Real Time PCR specific assays. Based on these results the most reliable and convenient method for UEs miRNAs extraction and analysis was selected. Advantages and drawbacks of each methodology were also discussed

Circadian exosomal expression of renal thiazide-sensitive NaCl cotransporter (NCC) and prostasin in healthy individuals

A circadian timing system is involved in the maintenance of fluid and electrolyte balance and blood pressure control. Aldosterone and vasopressin modulate ion transporters and channels crucial in sodium (Na) and water reabsorption such as the epithelium Na channel and the renal thiazide-sensitive NaCl cotransporter (NCC). We analyzed in urinary exosomes the intraday variations of NCC and prostasin expression and the association with electrolytes and water balance parameters