Differential Expression of Urinary Exosomal MicroRNAs in Type 2 Diabetic Kidney Disease

Background and aims: Diabetic kidney disease (DKD) is a progressive microvascular complication that leads to a decline in renal function and is the most frequent cause of end stage renal disease. Currently, there is a need for improved biomarkers for the early detection of DKD. MicroRNAs (miRNAs) are short, non-coding RNA molecules that modulate gene regulation that are commonly found in urinary exosomes and may reflect differential gene expression ongoing in the kidney during the disease process. We evaluated differential urinary exosomal miRNA expression in type 2 DKD (T2DKD) compared to control subjects with type 2 diabetes (T2D) and normal renal function.Materials and methods: Exosomes were harvested from 1.1 ml of cell-free urine according to the protocol for miRCURY Exosome Urine Kit (Qiagen). RNA was extracted using a miRCURY RNA Isolation Kit (Qiagen) and cDNA synthesised using a Universal cDNA Synthesis Kit (Qiagen). Differential urinary exosomal miRNA profiles were evaluated in 14 participants with T2DKD and 15 age and gender matched individuals with T2D and normal renal function using a miRCURY LNA miRNA Focus PCR Panel (Qiagen). The panels consisted of 87 miRNAs previously reported in human urinary exosomes. All miRNA expression was normalised using the reference genes selected by the NormFinder algorithm, using GenEx software for the analysis of quantitative PCR data. Statistical analyses were undertaken to compare the expression profiles between cases and controls using independent sample t tests and binary logistic regression to adjust for appropriate confounders (SPSS v.22).Results: Quantitative expression data was normalised according to the NormFinder algorithm using the 3 most stably expressed miRNAs in the panel (miR-200b-3p, 30c-5p and 27b-3p). Urinary miR-21-5p, 7e-5p and 23b-3p were significantly upregulated in T2DKD cases compared to T2D controls with good renal function (P<0.05). Conversely, miR-30b-5p and miR-125b-5p expression was significantly lower in T2DKD cases compared to T2D controls with normal renal function (P <0.05). In a binary logistic regression analyses adjusted for age, sex and mean arterial blood pressure, only miR- 21-5p remained significantly associated with T2DKD (odds ratio = 3.28, confidence intervals: 1.14 – 9.43; P=0.03), while miR-30b-5p, 7e-p 23b-3p and 125b-5p rose just above the established significance threshold (P>0.05). Conclusion: The results suggest miR-21-5p, 30b-5p, 125b-5p, 7e-5p and 23b-3p are differentially expressed in individuals with T2DKD, although only miR-21-5p remained significant in the fully adjusted model. Further independent validation and replication is ongoing to confirm the role of these miRNAs in T2DKD disease aetiology, and to evaluate their sensitivity as potential biomarkers of T2DKD.