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

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 (20–22 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™ 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™ 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™ 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™), 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® staining was obtained with the combination of TRI Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ 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™ 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™ 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.