Abstract
Objective: In a previous work we established an exosomal miRNA signature associated to urinary albumin excretion (UAE) in hypertension, which included microRNA (miRNA) miR-26a, and obtaining cell signaling (MAPK, calcium, p53), TGF-ß and VEGF pathways as the most regulated biological processes. The aim of this work was to validate the exosomal miR-26a changes and its potential association with TGF-ß in human podocytes. Design and method: This prospective study analysed the miR-26a levels in 52 hypertensive patients, 24 with UAE (162,8 ± 168.2 mg/g creatinine; mean age 52.7 ± 8.4 years) and 28 normoalbuminuric (mean age 54.5 ± 5.6 years). Exosomes were isolated by differential ultracentrifugation from urine and plasma and miRNA levels were calculated by qRT-PCR. Urinary levels of TGF-ß1 were quantified by ELISA. Finally, human podocytes cultures were stimulated with recombinant human TGF-ß1 at several concentrations (5 ng/mL, 10 ng/mL and 15 ng/mL) for up 24 h to assess miR-26a changes in exosomal and cellular fractions. Results: We found a decrease in the levels of miR-26-5p in plasma and urinary exosomes of patients con increased UAE (p < 0.05 and p < 0.01, respectively). In addition, miR-26a-5p correlated inversely with UAE levels, both in urinary and plasma exosomes (r = -0.49, p < 0.01; r = -0.53, p < 0.01). Our patients also showed a significant increase in urinary levels of TGF-ß1 (2-fold increase, p < 0.05) in hypertensive patients with UAE. Finally, we analysed the effect in vitro of TGF-ß1 stress. Significant changes in scaffolding of podocytes, such as an increase of vimentin levels, has been also observed. Finally, significant down-regulated miR-26a expression in exosomes from podocytes after TGF-ß1 stimulation (5 – 15 ng / mL): 2.23, 1.96 and 1.7-fold decrease, with p < 0.001 and p < 0.05, respectively. No significant changes were obtained at intracellular level. Conclusions: Our results show that miR-26a-5p is decreased in urinary and plasma exosomes of microalbuminuric patients. In addition, exosomal miR-26a podocyte release is associated with TGF-ß1 pathway, key molecule in the development and maintenance structure of the glomerular filtration barrier and fibrosis.
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