No abstract

Diabetic nephropathy is a major complication in diabetic patients. Podocytes undergo loss and detachment from the basal membrane. Intra- and intercellular communication through exosomes are key processes for maintaining function, and the Rab3A/Rab27A system is an important counterpart. Previously, we observed significant changes in the Rab3A/Rab27A system in podocytes under glucose overload, demonstrating its important role in podocyte injury. We investigated the implication of silencing the Rab3A/Rab27A system in high glucose-treated podocytes and analysed the effect on differentiation, apoptosis, cytoskeletal organisation, vesicle distribution, and microRNA expression in cells and exosomes. For this, we subjected podocytes to high glucose and transfection through siRNAs, and we isolated extracellular vesicles and performed western blotting, transmission electron microscopy, RT-qPCR, immunofluorescence and flow cytometry assays. We found that silencing RAB3A and RAB27A generally leads to a decrease in podocyte differentiation and cytoskeleton organization and an increase in apoptosis. Moreover, CD63-positive vesicles experienced a pattern distribution change. Under high glucose, Rab3A/Rab27A silencing ameliorates some of these detrimental processes, suggesting a differential influence depending on the presence or absence of cellular stress. We also observed substantial expression changes in miRNAs that were relevant in diabetic nephropathy upon silencing and glucose treatment. Our findings highlight the Rab3A/Rab27A system as a key participant in podocyte injury and vesicular traffic regulation in diabetic nephropathy.

Objective: Endothelial dysfunction is a forerunner of atherosclerosis, leading to cardiovascular disease, and albuminuria is a marker of endothelial dysfunction. Measurement of circulating levels of microRNAs is emerging as potential biomarkers for cardiovascular disease. We aimed to estimate the predictive value of a plasma microRNAs signature associated with albuminuria in the incidence of cardiovascular events. Design and method: The first study phase was to identify the plasma miRNAs associated with albuminuria in patients with HTN and controls. In a second step, a predictive model of miRNAs to assess the risk to develop CV events was analyzed in a retrospective study of the general population (HORTEGA Follow-up Study), on those who did not have previous CVD and had complete basal information on relevant covariates. Plasma miRNAs quantified in hypertensive patients by next generation sequencing were validated in a cohort of patients and controls by real-time quantitative PCR. The microRNAs associated with albuminuria were tested and their prognostic value for cardiovascular event incidence were analyzed on a retrospective, population-based study using Cox proportional hazard models. Results: A plasma microRNA profile was identified in the discovery cohort (n = 48) associated with albuminuria and three microRNAs (miR-126-3p, miR-1260b and miR-374a-5p) were confirmed in the validation cohort (n = 98). The microRNA signature discriminates urinary albumin excretion at baseline (n = 1025), and predicts the incidence of cardiovascular events and coronary heart disease and stroke in a general population retrospective study within a 14-year follow-up (n = 926). Thus, high miR-126-3p levels were associated with a shorter time free of both cardiovascular events (HR = 1.48, (1.36–1.62), p < 0.0001), as well as coronary artery disease and stroke combined (HR = 2.49, (2.19–2.83), p < 0.0001) (Figure). Conclusions: An increased plasma microRNAs profile was identified in hypertensive patients with albuminuria. Increased miR-126-3p emerges as a prognostic marker for cardiovascular events in a long-term general population. Further studies will assess the potential role of miR-126-3p as a guide for the status of endothelial dysfunction.