Urinary- and Plasma-Derived Exosomes Reveal a Distinct MicroRNA Signature Associated With Albuminuria in Hypertension.

Urinary albumin excretion (UAE) is a marker of cardiovascular risk and renal damage in hypertension. MicroRNAs (miRNAs) packaged into exosomes function as paracrine effectors in cell communication and the kidney is not exempt. This study aimed to state an exosomal miRNA profile/signature associated to hypertension with increased UAE and the impact of profibrotic TGF-ß1 (transforming growth factor ß1) on exosomes miRNA release. Therefore, exosomes samples from patients with hypertension with/without UAE were isolated and characterized. Three individual and unique small RNA libraries from each subject were prepared (total plasma, urinary, and plasma-derived exosomes) for next-generation sequencing profiling. Differentially expressed miRNAs were over-represented in Kyoto Encyclopedia of Genes and Genomes pathways, and selected miRNAs were validated by real-time quantitative polymerase chain reaction in a confirmation cohort. Thus, a signature of 29 dysregulated circulating miRNAs was identified in UAE hypertensive subjects, regulating 21 pathways. Moreover, changes in the levels of 4 exosomes-miRNAs were validated in a confirmation cohort and found associated with albuminuria. In particular miR-26a, major regulator of TGF-ß signaling, was found downregulated in both type of exosomes when compared with healthy controls and to hypertension normoalbuminurics (P<0.01). Similarly, decreased miR-26a levels were found in podocyte-derived exosomes after TGF-ß stress. Our results revealed an exosomes miRNA signature associated to albuminuria in hypertension. In particular, exosomes miR-26a seemed to play a key role in the regulation of TGF-ß, a relevant effector in podocyte damage. These findings support the use of exosomes miRNAs as biomarkers of cardiovascular risk progression and therapeutic tools in early kidney damage.

Decreased Urinary Levels of SIRT1 as Non-Invasive Biomarker of Early Renal Damage in Hypertension.

Sirtuins have become important players in renal damage in hypertension and diabetes, but their value as biomarkers is poorly assessed. The aims of the study were to evaluate the levels of sirtuin1 (SIRT1), and two miRNAs that regulate SIRT1 expression in hypertensive patients with incipient renal damage with and without diabetes. We quantified urinary SIRT1 and claudin 1 (CLDN1) mRNA and miR34-a and miR-200a levels by quantitative real-time polymerase chain reaction (RT-qPCR) from patients and in cultured podocytes treated with high glucose and angiotensin II. Western blot and fluorescence analyses were also performed. We found decreased SIRT1 levels in patients with increased urinary albumin excretion (UAE), the lowest with diabetes presence, and a strong association with UAE, discriminating incipient renal damage. In vitro experiments also showed SIRT1 overall decreases in podocyte cultures under treatment conditions. In urine samples, miR-34a was reduced and miR-200a increased, both related to UAE levels. However, both miRNAs were generally increased in podocyte cultures under high glucose and angiotensin-II treatment. These results show a significant urinary SIRT1 decrease in albuminuric hypertensive patients, strongly associated with albuminuria, suggesting that SIRT1 could be a potential and non-invasive method to assess incipient renal damage in hypertensive patients.

Cardiac magnetic resonance-derived fibrosis, strain and molecular biomarkers of fibrosis in hypertensive heart disease.

AIMS: Myocardial fibrosis is a relevant component of hypertensive heart disease (HHD). Novel cardiovascular magnetic resonance (CMR) imaging techniques have shown potential in quantification of diffuse cardiac fibrosis, with T1 mapping, and estimating preclinical cardiac dysfunction, with strain analysis. Molecular biomarkers of fibrosis have been related with clinical outcomes and histologically proven myocardial fibrosis. The relationship between these CMR-imaging techniques and circulating biomarkers is not fully understood. METHODS AND RESULTS: CMR was performed on a 3T scanner in 36 individuals with HHD. Extracellular volume fraction (ECV) and the partition coefficient were assessed using the T1 mapping technique shMOLLI. Longitudinal, circumferential and radial strain was assessed using CMR-Feature Tracking. Molecular biomarkers of collagen synthesis (PICP and PIIINP) and collagen degradation (CITP and MMP-1) were measured in blood using commercial kits. Correlation models showed a significant relationship of T1 mapping measures with left atrial diameter, LV mass, LV posterior wall thickness, LV end-diastolic volume and longitudinal strain. In fully adjusted regression models, ECV was associated with left atrial diameter (ß=0.75, P = 0.005) and longitudinal strain (ß = 0.43, P = 0.030); the partition coefficient was associated with LV posterior wall thickness (ß = 0.53, P = 0.046). Strain measures were associated with cardiac geometry, and longitudinal strain was marginally associated with CITP. CONCLUSION: In individuals with HHD, CMR-derived measures of myocardial fibrosis and function are related and might be useful tools for the identification and characterization of preclinical cardiac dysfunction and diffuse myocardial fibrosis. Molecular biomarkers of fibrosis were marginally associated with myocardial strain, but not with the extension of CMR-measured cardiac fibrosis.

Urinary exosome miR-146a is a potential marker of albuminuria in essential hypertension.

BACKGROUND: There is increasing interest in using extracellular vesicle-derived microRNAs (miRNAs) as biomarkers in renal dysfunction and injury. Preliminary evidence indicates that miRNAs regulate the progression of glomerular disease. Indeed, exosomes from the renal system have provided novel evidence in the clinical setting of albuminuria. Thus, the aim of this study was to quantify the urinary miRNAs present in exosome and microvesicles (MVs), and to assess their association with the presence of increased urinary albumin excretion in essential hypertension. METHODS: Exosomes were collected from urine specimens from a cohort of hypertensive patients with (n = 24) or without albuminuria (n = 28), and from 20 healthy volunteers as a control group. Urinary exosomes were phenotyped by Western blot, tunable resistive pulse sensing, and electronic microscopy. Expression of miR-146a and miR-335* was analysed by qRT-PCR and any associations between albuminuria and exosomal miRNAs were analysed. RESULTS: Urinary miRNAs are highly enriched in exosome subpopulations compared to MVs, both in patients with or without increased albuminuria (p < 0.001), but not in the control group. High albuminuria was associated with 2.5-fold less miR-146a in exosomes (p = 0.017), whereas miR-146a levels in MV did not change. In addition, exosome miR-146a levels were inversely associated with albuminuria (r = 0.65, p < 0.0001), and discriminated the presence of urinary albumin excretion presence [area under the curve = 0.80, 95% confidence interval: 0.66-0.95; p = 0.0013]. CONCLUSIONS: Our results indicate that miRNAs were enriched in the urinary exosome subpopulation in hypertensive patients and that low miR-146a expression in exosomes was associated with the presence of albuminuria. Thus, urinary exosome miR-146a may be a potentially useful tool for studying early renal injury in hypertension.

Urinary podocyte-associated molecules and albuminuria in hypertension.

OBJECTIVE: Hypertension-induced podocyte damage and the relationship with UAE is analyzed in diabetic and nondiabetic participants. PATIENTS AND METHODS: Sixty-four hypertensive patients, 30 diabetics, with glomerular filtration rate (eGFR) greater than 60 ml/min per 1.73 m were included. Urinary albumin excretion was measured in morning urine using a nephelometric immunoassay and expressed as albumin/creatinine ratio. Urinary pellets were obtained from fresh urine and mRNA was assessed by real-time quantitative PCR. Likewise, protein podocyte-specific molecules were measured by western blot using specific antibodies. RESULTS: Fourteen nondiabetics and 20 diabetics had increased UAE greater than 30 mg/g. In individuals with increased EUA, the mRNA expression of nephrin and CD2AP was low in diabetics, whereas only nephrin mRNA in nondiabetics. No differences were observed in podocalyxin and aquaporin-1 mRNA levels. Concerning the protein values, in both nondiabetic and diabetic patients, nephrin, CD2AP and podocalyxin were increased in patients with increased UAE, with no differences in aquaporin-1. A significant positive relationship was observed between log UAE and nephrin protein values, and an inverse association observed with mRNA. CONCLUSION: Hypertensive patients who had elevated UAE showed increased urinary excretion of podocyte-specific proteins coupled with a phenotype of decreased mRNA expression. The phenotype of podocyte-specific mRNA and the increment of nephrin can be used as a valuable marker of early glomerular injury.

Pulse pressure amplification and its determinants.

BACKGROUND: Pulse pressure (PP) amplification expressed as the peripheral-to-central PP ratio has gained importance in the assessment of cardiovascular phenotypes and cardiovascular risk. The aim of the present study was to assess the relationship between PP amplification, large vessel parameters and peripheral blood pressure (BP) to gain insights into the amplification phenomenon. METHODS: Peripheral BP, central BP and carotid-femoral pulse wave velocity (cfPWV) were assessed using the OMRON M6, SphygmoCor and Complior devices, respectively, in 741 adults attending the hypertension outpatient clinic. Analysis of covariance, partial correlations and multiple linear regression models were performed to assess the relationship between PP amplification, peripheral BP and cfPWV. RESULTS: PP amplification was inversely related to BP group. Women showed lower PP amplification than men (1.24 ± 0.18 and 1.35 ± 0.18, respectively, p < 0.001). Age, female gender and mean arterial pressure were inversely associated with PP amplification (p < 0.001), whereas heart rate and body mass index showed positive associations (p < 0.001 and p = 0.049, respectively). cfPWV was a predictor of PP amplification in men but not in women (p = 0.006 and p = 0.424, respectively). CONCLUSIONS: PP amplification is related to BP: the higher the BP, the lower the PP amplification. Gender, age and body composition have a significant impact on PP amplification.

Carotid-femoral pulse wave velocity assessment by two different methods: implications for risk assessment.

INTRODUCTION: Several devices are available for carotid-femoral pulse wave velocity (cfPWV) measurement, and a cut-off value for reference cfPWV has been established. However, discrepancies between devices have been reported. OBJECTIVES: The aim of the study was to establish the concordance of two common techniques (Complior and SphygmoCor), taking into account the anatomical distance between the measurement sites, and to investigate the impact on cardiovascular risk stratification. METHODS: cfPWV, central and peripheral blood pressure were assessed in patients attending the hypertension outpatient clinic. The subtracted carotid-femoral distance was estimated both according to the manufacturer's recommendations and correcting the obtained values by 10.3%. Bland-Altman plots, Pearson's correlation coefficient, Lin's concordance correlation coefficient and multivariate models were used to investigate the difference in cfPWV. RESULTS: cfPWV assessed in 118 patients (age 55 ±â€Š12 years, 61% hypertensive patients, BMI 28.9 ±â€Š4.4  kg/m2) with the Complior device was lower than that assessed with the SphygmoCor device, regardless of correcting the subtracted carotid-femoral distance (8.7 vs. 10.3  m/s and 9.3  m/s, respectively; P value < 0.001). The average difference was -1.59 ±â€Š1.5 and -0.617 ±â€Š1.39  m/s for corrected and uncorrected SphygmoCor values, respectively, SBP, BMI and female being the main determinants of the difference. Cardiovascular risk stratification changed in up to 40% of the study population, depending on the device and the arterial distance estimation. CONCLUSION: The concordance between the Complior and the SphygmoCor device is poor when the anatomical artery length is controlled for and in the presence of cardiovascular risk factors, resulting in a difference in classification of cardiovascular risk.

Influence of obesity in central blood pressure.

BACKGROUND: Obesity is an important risk factor for cardiovascular disease and has become a major concern in healthcare due to its high prevalence worldwide. The aim of the present study was to investigate the impact of BMI on central blood pressure (BP) and pulse wave velocity (PWV) in normotensive and hypertensive patients. PATIENTS AND METHODS: Normotensive and hypertensive adult patients who attended the outpatient clinic of cardiovascular risk were included. Peripheral BP was obtained in the brachial artery by using an oscillometric device (OMRON M-6). Central aortic BP waveform was reconstructed from the radial artery pressure waveforms (SphygmoCor, AtCor Medical, Sydney, Australia) and central BP was calculated. Carotid-femoral PWV was measured by an automatic device (Complior, Artech, France). RESULTS: We examined a total of 351 patients [50.7% women; 77 patients normal-weight (BMI < 25 kg/m)], 274 patients overweight or obese (BMI ≥25 kg/m). Central SBP showed a positive association with male sex and mean BP, but a negative association with overweight/obesity. PWV was positively associated with age, male sex, central BP, peripheral BP and BP treatment, whereas BMI of at least 25 kg/m led to a decrease in PWV in patients with the same central SBP levels. Likewise, PWV was lower in the overweight/obese group compared to the normal-weight group at the same central SBP. CONCLUSION: Overweight and obesity tend to have lower central SBP as compared to lean patients, mainly in women. Further research is required to assess the interaction between body weight and vascular dynamics and their clinical implications.