The Repaire Mechanism of Mesenchymal Stem Cell microvesicle miR‐451a in Diabetic Nephropathy
Authors: Ling Zhong 
Published: November 2023

Objective

Mescenchymal stem cells (MSCs) play a central role in the repair of cell and tissue damage. Microvesicles (MVs) derived from MSCs have been described as a new mechanism of cell‐to‐cell communication. Furthermore, MVs shuttle a specific subset of cellular miRNA. miR‐451a was highly expressed in MVs. miR‐451a regulates the F‐box protein family invoved in Diabetic Nephropathy (DN) focused on signal transduction and cell cycle. Epithelial to mesenchymal transition (EMT) was critical for the renal fibrosis in Diabetic Nephropathy (DN). We studied the protective effects and mechanisms of miR‐451a in fibrosis of DN. In this study, the effects of MVs‐miR‐451a on EMT and fibrosis of DN were evaluated.

Methods

MVs were obtained from MSCs supernatant by gradient centrifugation and ultracentrifugation. MVs were identified by scanning electron microscope, particle size analyzer, and flow cytometry. RNAs was extracted from MSC‐MVs. miR‐451a level in MSCs and MVs were tested through fluorogenic quantitative PCR (qPCR). HK‐2 damage model is established by the high sugar (30 mM) and uric acid (10 mg/dL) in vitro. MVs, miR‐451a mimic, miR‐451a inhibitor were added respectively to study change of miR‐451a's level, p15INK4b (p15) and p19INK4d (p19)'s level (represented cell cycle), E‐cadherin and α‐SMA (represented fibrosis) by qPCR. The corresponding protein expression levels of the markers were determined by WB. The changes of cell cycle were analyzed by flow cytometry. The cell proliferation ability and viability were tested by BrdU and CCK8. The mannitol hypertonic and fibroblasts‐micro capsule (HFF‐MV) control groups were set up.

Constructed plasmids target genes p19INK4d 3 ′UTR region wild type plasmid and mutation plasmid. Determined p19INK4d was one of the miR‐451a target genes and the binding sites by dual luciferase reaction. The MVs and miR‐451a agomir were used to treat renal injury of fibrosis induced by high glucose and uric acid in vivo. The markers were the same to study in vitro. Furthermore, the renal function of mice was tested.

Results

MVs showed the presence of several markers known to be expressed on MSCs. The size of MVs ranged from 10 nm to 1μm. MVs enriched miR‐451a (4.87 fold change of MSCs). The expression level of miR‐451a in injured HK2 cell and kidney were decreased to 0.28 and 0.067 (compared with the control). Infusion of MVs and miR‐451a reduced HK2 cell and mice renal genes and proteins expression of p15, p19 and the factors associate with fibrosis. And they up‐regulated the proportion of S and G2, and stimulated the proliferation and viability of HK2 cell. p19 was identified as one of target gene of miR451a. After transplantation of MVs and agomir into DN mice with high uric acid, improvements in renal function and histopathological change of kidney were observed. Engrafted MVs were localized to areas of injured kidney.

Conclusion

Our results indicate that infused MVs may improve the recovery from fibrosis in DN mice with high uric acid by increasing miR‐451a expression to re‐start cell proliferation and regulate EMT.