Glomerular mesangial derived extracellular vesicles deteriorate diabetic kidney disease via miR-3147/PRKAR2B axis

Figures

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  Figure 1.. Elevated extracellular vesicles (EVs) derived miR-3147 exhibited diagnostic value and clinical correlation in diabetic kidney diseases (DKD). A. Volcano plotting of the differentially expressed miRNAs (De-miRNAs) between DKD, diabetes mellitus (DM) and healthy control; B. Heatmap illustrated the pattern of 15 De-miRNAs, including 6 upregulated and 9 downregulated; C. qPCR verification of the expression of above 15 De-miRNAs. miR-16-5p, miR-615-3p and miR-3147 were confirmed with significant changes and miR-3147 with lowest p-value was selected for further experiments. D. EV derived miR-3147 diagnostic efficacy differentiating DKD from DM (AUC = 0.80, specificity = 0.94, sensitivity = 0.59).

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  Figure 2.. Correlations of significance between miR-3147 levels and clinical parameters of diabetic kidney diseases (DKD) patients. Significant correlations were found between miR-3147 level and age, eGFR-MDRD, eGFR-CKD-EPI, blood urea nitrogen (BUN), creatinine, protein-to-creatinine ratio, HbA1c, T2DM duration and serum cystatin-C.

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  Figure 3.. Localization and expression evaluation of miR-3147 in kidney biopsy from diabetic kidney diseases (DKD) patients and normal subjects. Localization of miR-3147 in mesangial cells (MCs) was confirmed by the co-staining of the MC marker thy-1.1 and increased level of miR-3147 was found in DKD patients compared to normal controls. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. normal controls.

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  Figure 4.. Increased level of miR-3147 after high glucose (HG) challenge and its overexpression could result in mesangial cells (MCs) hyper-proliferation. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. HG group at 48 h. LG: low glucose.

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  Figure 5.. High glucose (HG) induced miR-3147 overexpression could result in mesangial cells (MCs) apoptosis. A. TUNEL staining confirmed that miR-3147 overexpression could increase cell apoptosis in HG treated MCs, whereas miR-3147 inhibition abolished these effects. B. TUNEL staining fluorescence quantification. C. Flow cytometry annexin V-PI staining revealed the effects exerted by miR-3147 was mainly on early apoptosis. D. Early apoptosis quantification. E. Late apoptosis quantification. *p < 0.05 and **p < 0.01 vs. HG group. LG: low glucose.

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  Figure 6.. miR-3147 exerted its effects on high glucose (HG) treated mesangial cells (MCs) via targeting PRKAR2B. A. Illustration of interaction between target PRKAR2B and miR-3147; B. Dual luciferase activity analysis of Wild type (WT) and Mutated (MUT) PRKAR2B confirmed its interaction with miR-3147. C-D. MiR-3147 overexpression could decrease PRKAR2B mRNA (C) and protein levels (D) by qPCR in high glucose treated MCs, while miR-3147 inhibition could reverse this effects. *p < 0.05 and **p < 0.01 vs. HG group. LG: low glucose.

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  Figure 7.. PRKAR2B reversed the effect of miR-3147 overexpression on early apoptosis on mesangial cells (MCs) in HG condition. A. Flow cytometry annexin V-PI staining confirmed that miR-3147 overexpression increased early apoptosis in HG treated MCs, whereas PRKAR2B overexpression reversed these effects. B. Early apoptosis quantification. C. Late apoptosis quantification. *p < 0.05, **p < 0.01 and ***p < 0.001 for between group comparison.