RESUMO
Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.
RESUMO
OBJECTIVES: To define the patterns and causes of hearing decline associated to Turner's syndrome (TS). METHODS: An observational study with three cohorts was designed: 31 TS patients, 15 women with other congenital hypogonadims (OCH) and 41 healthy age-matched women taking contraception. Microotoscopy, standard pure-tone audiometry brain auditory evoked potentials (BAEP) were performed to study hearing function. RESULTS: Up to 87% of TS subjects suffered from some degree of hearing loss (HL) in the audiograms, compared with 20% OCH and 27% controls. Sensorineural hearing loss (SNHL) was the most frequent type of hypoacusia found in TS group. BAEP study demonstrated that 61% of TS women showed HL compared to 20% in OCH patients. No significant differences in latencies, amplitudes, and interpeaks of waves I, III and V were found between TS and OCH, nor when compared to reference population. Worse results were observed among the oldest TS patients, those with pure monosomy or isochromosome, and those with a history of recurrent otitis. CONCLUSIONS: More than a half of TS females presented HL. SNHL is the most frequent pattern among middle-aged women with TS. Old age, karyotype and recurrent otitis are predisposition factors to produce HL, while oestrogens play a minor role.