Renal Tissue-Derived Exosomal miRNA-34a in Diabetic Nephropathy Induces Renal Tubular Cell Fibrosis by Promoting the Polarization of M1 Macrophages

Abstract

Background: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease, and the activation and infiltration of phagocytes are critical steps of DN. This study aimed to explore the mechanism of exosomes in macrophages and diabetes nephropathy and the role of miRNA-34a, which might provide a new path for treating DN.

Materials and methods: The DN model was established, and the success of the model establishment was confirmed by detecting general indicators, HE staining, and immunohistochemistry. Electron microscopy and NanoSight Tracking Analysis (NTA) were used to see the morphology and size of exosomes. MiRNA-34a inhibitor, miRNA-34a mimics, pc-PPARGC1A, and controls were transfected in macrophages with or without kidney exosomal. A dual-luciferase reporter gene experiment verifies the targeting relationship between miRNA-34a and PPARGC1A. After exosomal culture, macrophages are co-cultured with normal renal tubular cells to detect renal tubular cell fibrosis. Q-PCR and western blot were undertaken to detect related RNA and proteins.

Results: An animal model of diabetic nephropathy was successfully constructed. Macrophages could phagocytose exosomes. After ingesting model exosomes, M1 macrophages were activated, while M2 macrophages were weakened, indicating the model mice's kidney exosomes caused the polarization. MiRNA-34a inhibitor increased PPARGC1A expression. MiRNA-34a expressed higher in diabetic nephropathy Model-Exo. MiRNA-34a negatively regulated PPARGC1A. PPARGC1A rescued macrophage polarization and renal tubular cell fibrosis.

Conclusion: Exosomal miRNA-34a of tubular epithelial cells promoted M1 macrophage activation in diabetic nephropathy via negatively regulating PPARGC1A expression, which may provide a new direction for further exploration of DN treatment.

Figure 1

A mouse model of diabetic nephropathy was successfully constructed: (a) blood glucose concentration, (b) the weight of the mouse in the model and control groups, (c) BUN concentration in serum, (d) Scr concentration in serum, (e) content of urine uric acid, and (f) HE staining and F4/80 immunohistochemical assay.  ^∗∗P < 0.01,  ^∗∗∗P < 0.001vs. Ctrl group.

Figure 2

Extraction and identification of kidney exosomes in model mice and miRNA detection: (a) morphology of kidney exosomes under electron microscope, (b) nanoparticle tracking analysis of exosomes, (c) expression of exosomal markers (TSG101, Grp94, CD63), and (d) Q-PCR detection of miRNA-34a in kidney exosomes.  ^∗∗∗P < 0.001 vs. Ctrl-Exo group.

Figure 3

The kidney exosomes of model mice caused the polarization of macrophages: (a) macrophages take up PKH26-labeled kidney exosomes, (b) macrophage miRNA-34a expression after uptake of exosomes, (c) immunofluorescence method was processed to observe the expression of iNOS and Arg1, (d) expression of macrophage M1 phenotypic markers (iNOS and CD86), (e) expression of macrophage M2 phenotypic markers (CD206 and Arg-1), and (f) western blot detection was undertaken to detect macrophage M1 and M2 phenotypic markers.  ^∗∗∗P < 0.001vs. Ctrl-Exo group.

Figure 4

Macrophages treated with model exosomes promote renal tubular cell TCMK-1 fibrosis: (a) schematic diagram of cocultivation, (b) western blot detection for fibrosis-related indicators (FN/α-SMA/Collagen I), and (c) immunofluorescence detection for fibrosis-related indicators (FN/α-SMA/Collagen I).  ^∗∗∗P < 0.001vs. M^Ctrl-Exo group.

Figure 5

miRNA-34a-targeted PPARGC1A: (a) the binding site between PPARGC1A and miRNA-34a, (b) miRNA-34a mimic significantly decreased the expression of PPARGC1A, (c) miRNA-34a inhibitor obviously increased PPARGC1A expression, and (d) Q-PCR detection for the relationship between PPARGC1A and miRNA-34a expression.  ^∗∗P < 0.01,  ^∗∗∗P < 0.001vs. NC inhibitor group.

Figure 6

pc-PPARGC1A rescued macrophage polarization: (a) the immunofluorescence method was used to observe the expression of iNOS and Arg1, (b) expression of macrophage M1 phenotypic markers (iNOS and CD86), (c) expression of macrophage M2 phenotypic markers (CD206 and Arg-1), and (d) western blot detection was undertaken to detect macrophage M1 and M2 phenotypic markers. Compared with Ctrl-Exo group,  ^∗∗∗P < 0.001, compared with Model-Exo+pc-NC group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Figure 7

pc-PPARGC1A rescued the effect of macrophages on renal tubular cell fibrosis: (a) western blot detection for fibrosis-related indicators (FN/α-SMA/Collagen I) and (b) immunofluorescence detection for fibrosis-related indicators (FN/α-SMA/Collagen I). Compared with Ctrl-Exo group,  ^∗∗∗P < 0.001, compared with Model-Eexo+pc-NC group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.