Urinary Exosomal MicroRNAs in Incipient Diabetic Nephropathy

<div><p>MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication. </p> </div

Expression of miR-145 in experimental diabetes.

<p>MiR-145 levels were measured by qRT-PCR in both urinary exosomes (<b>A</b>) and isolated glomeruli (<b>B</b>) from diabetic (DM) and control (ND) mice as described in the Methods. Results, corrected for the expression of housekeeping U6 snRNA, are shown in the graphs (*p<0.001 DM vs ND). </p

Expression of miR-145 in human mesangial cells.

<p>(<b>A</b>) Expression of STAT-1, a known miR-145 target, was assessed by immunoblotting in total protein extracts from mesangial cells transfected with either miR-145 mimic or scramble oligonucleotides. Tubulin was used as loading control. Results are depicted in the graph (*p<0.05) and a representative immunoblot is shown. (<b>B</b>) Human mesangial cells were exposed to increasing (6.8, 15, and 25 mM) glucose concentrations for 4,6,12 and 24 hours. Total RNA was extracted and levels of miR-145 measured by qRT-PCR (*p<0.01) 15 and 25 mM glucose vs. 6.8 mM glucose at all time points). (<b>C</b>) Human mesangial cells were exposed to either normal (6.8 mM-NG) or high (25 mM-HG) glucose concentrations for 24 hours. Exosomes were isolated from the supernatants by ultracentrifugation as described in the Methods. Total RNA was extracted and levels of miR-145 measured by qRT-PCR (*p<0.001 HG vs. NG).</p