LncTUG1 ameliorates renal tubular fibrosis in experimental diabetic nephropathy through the miR-145-5p/dual-specificity phosphatase 6 axis

Abstract

The renal interstitial fibrosis contributes to the progression and deterioration of diabetic nephropathy (DN). Long noncoding RNA taurine-up-regulated gene 1 (TUG1) in kidneys may be down-regulated by hyperglycemia. We aim to explore its role in tubular fibrosis caused by high glucose and the possible target genes of TUG1. In this study, a streptozocin-induced accelerated DN mouse model and a high glucose-stimulated HK-2 cells model was established to evaluate TUG1 expression. Potential targets of TUG1 were analyzed by online tools and confirmed by luciferase assay. A rescue experiment and gene silencing assay were used to investigate whether TUG1 plays its regulation role via miR-145-5p/dual-specificity phosphatase 6 (DUSP6) in HK2 cells. The effects of TUG1 on inflammation and fibrosis in high glucose treated tubular cells were evaluated by in vitro study, as well as in vivo DN mice model through AAV-TUG1 delivery. Results showed TUG1was downregulated in HK2 cells incubated with high glucose while miR-145-5p was upregulated. Overexpression of TUG1 alleviated renal injury by suppressing inflammation and fibrosis in vivo. Overexpression of TUG1 inhibited HK-2 cell fibrosis and relieved the inflammation. A mechanism study demonstrated that TUG1 directly sponged to miR-145-5p, and DUSP6 was identified as a target downstream of miR-145-5p. In addition, miR-145-5 overexpression and DUSP6 inhibition countervailed the impacts of TUG1. Our findings revealed that TUG1 overexpression alleviates kidney injury in DN mice and decreases the inflammatory response and fibrosis of high glucose-stimulated HK-2 cells via miR-145-5p/DUSP6 axis.

Keywords: DUSP6; Diabetic nephropathy; inflammation; interstitial fibrosis; lncTUG1; miR-145-5p.

Figure 1.

mRNA expressions or protein levels of TUG1, DUSP6, and fibrosis-related genes in HK2 cells treated by normal glucose (NG) and high glucose (HG), and kidney tissues from experimental diabetic nephropathy mice. (A) TUG1 mRNA; (B) DUSP6 mRNA; (C) collagen IV (COL4A2) mRNA; (D) fibronectin (FN1) mRNA. (E) protein levels of fibronectin and collagen IV; (F) BUN and Scr in DN mice. (G) Tubular injury and fibrosis by HE and Masson’s trichrome in DN mice; (H) quantitative analysis of tubular injury and fibrosis; (I) mRNA expression of TUG1 and DUSP6 in kidney tissues from DN and normal control mice. **p < 0.01.

Figure 2.

Delivery of AAV-TUG1 ameliorates kidney injuries in DN mice. (A) TUG1 expression increased significantly in kidney tissues in mice by AAV-mediated gene delivery; (B–C) AAV-TUG1 overexpression significantly attenuates the pathological injuries (HE staining analysis) and fibrosis (Masson’s trichrome) in DN mice. (D–E) AAV-TUG1 overexpression significantly attenuates the renal TGF-β1, collagen I and III protein level in DN mice by immunohistochemistry. (F) AAV-TUG1 overexpression significantly improves kidney function by decreasing BUN and Scr concentration; (G) AAV-TUG1 overexpression attenuates mRNA levels of TNFα and IL-6 in DN mice; (H) AAV-TUG1 overexpression attenuates protein levels of TNFα and IL-6 in DN mice.

Figure 3.

Over-expression of TUG1 reduces proinflammation and profibrotic caused by high glucose toxicity in HK2 cells. (A) Relative expression of TUG1 increased after TUG1 transfection; (B) mRNA levels of TNF-α and IL-6 in HG-treated HK2 cells were significantly reduced by overexpression of TUG1; (C) mRNA levels of fibronectin (FN1) and collagen IV (COL4A2) in HG-treated HK2 cells were significantly reduced by overexpression of TUG1; (D) protein levels of fibronectin, collgen IV, TNF-α and IL-6 by Western blot. **p < 0.01.

Figure 4.

Inhibition of miR-145-5p alleviates tubular fibrosis under HG condition. (A) miR-145-5p is the target gene of TUG1. The potential binding sites between miR-145-5p and TUG1 were analyzed by online target tools; (B) MiR-145-5p expression is up-regulated in DN patients by GSE dataset GSE51647 and GSE 114477. (C) MiR-145-5p expression in HK2 cells increased after HG stimulation; (D) the binding between miR-145-5p to TUG1 was verified by the luciferase reporter assay. **p < 0.01.

Figure 5.

Inhibition of miR-145-55 significantly reduces the fibrosis characteristics in HG-treated HK2 cells. (A) Expression of miR-145-5p was significantly reduced by its inhibitor transfection. (B) TUG1 expression increased in NG and HG culture after miR-145-5p was inhibited; (C) cell viability of HK2 of miR-145-5p NC and miR-145-5p inhibitor under HG condition; (D) protein expressions of DUSP6, Collagen IV and fibronectin in miR-145-5p NC and miR-145-5p inhibitor group under HG condition.

Figure 6.

DUSP6 is the target gene of miR-145-5p. (A) potential targets of miR-145-5p were analyzed by online target tools; B the possible binding site of miR-145-5p to DUSP6 by bioinformatics prediction (C) Interaction between DUSP6 and miR-145-5p is verified by the luciferase reporter assay; (D) HK2 cell viability increased by miR-145-5p inhibition under HG condition, but was effectively abolished by DUSP6 knocking down. **p < 0.01.

Figure 7.

Knocking down DUSP6 abolishes the TUG1 effect on reducing fibrosis in HG-treated HK2 cells. (A) DUSP6 was knocked down in TUG1 stable expressed HK2 cells; (B–C) Reducing collagen IV and fibronectin ability of TUG1 was abolished by silencing DUSP6 in HG-treated HK2 cells confirmed by western blot and quantitative analysis. **p < 0.01.