Triptolide Attenuates Renal Tubular Epithelial-mesenchymal Transition Via the MiR-188-5p-mediated PI3K/AKT Pathway in Diabetic Kidney Disease

Abstract

Triptolide possesses the trait of renal protection. Epithelial-mesenchymal transition (EMT) is closely linked to the pathogenesis of diabetic kidney disease (DKD). MicroRNAs have recently emerged as critical regulators of DKD. However, it is poorly understood whether triptolide alleviates renal EMT by regulating microRNAs in DKD. In this study, we found that triptolide decreased albuminuria, improved the renal structure and reduced renal EMT in rats with DKD. Furthermore, activation of the PI3K/AKT signaling pathway was increased in diabetic rats, which was partly reversed by triptolide. Triptolide also alleviated glucose-induced EMT in HK-2 cells in vitro. PI3K/AKT signaling pathway activation was reduced after triptolide treatment. Moreover, triptolide decreased the increase in miR-188-5p expression stimulated by high glucose levels in HK-2 cells. miR-188-5p inhibited PTEN expression by directly interacting with the PTEN 3'-untranslated region. Additionally, downregulation of miR-188-5p, which imitates the effects of triptolide, attenuated the activation of the PI3K/AKT pathway and HG-induced EMT, whereas miR-188-5p overexpression reversed the effects of triptolide on the PI3K/AKT pathway and EMT. In conclusion, we demonstrated that triptolide ameliorates renal EMT via the PI3K/AKT signaling pathway through the interaction between miR-188-5p and PTEN, indicating that miR-188-5p may be a therapeutic target of triptolide in DKD.

Keywords: Diabetic kidney disease.; Epithelial-mesenchymal transition; MiR-188-5p; PTEN; Triptolide.

Figure 1

Renal pathological changes in animal subjects. Representative images of hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson's trichrome (Masson) stained kidney sections (inset images indicate augmentative renal tubules). Original magnification is ×400. The scale bar represents 100 μm. NC: normal control; DKD: diabetic kidney disease; TP: triptolide.

Figure 2

Triptolide reduced renal EMT and inactivated the PI3K/AKT signaling pathway in vivo. (A) Representative images of E-cadherin, vimentin and α-SMA by immunohistochemistry from renal tubules. Original magnification is ×400. The scale bar represents 50 μm. (B) Representative E-cadherin, vimentin and α-SMA bands by Western blot in rat kidneys. (C) Densitometric analysis of E-cadherin, vimentin and α-SMA by Western blot (n=5). (D) Representative PTEN, PI3K, p-AKT and t-AKT bands by Western blot in rat kidneys. (E) Densitometric analysis of PTEN, PI3K, p-AKT and t-AKT by Western Blot (n=5). Data are expressed as the mean ± SD. *P < 0.05 vs. the NC group. #P < 0.05 vs. the DKD group. NC: normal control; DKD: diabetic kidney disease; TP: triptolide.

Figure 3

Triptolide modulated the expression of E-cadherin, vimentin and α-SMA by inactivating the PI3K/AKT signaling pathway in HK-2 cells. (A) Representative images of E-cadherin (green), vimentin (green) and α-SMA (green) by immunofluorescence in HK-2 cells. Blue staining refers to the DAPI-stained nuclei. Original magnification is ×200. (B) Quantification of E-cadherin, vimentin and α-SMA gene expression in HK-2 cells (n=4). (C) Representative E-cadherin, vimentin and α-SMA bands by Western blot in HK-2 cells. (D) Densitometric analysis of E-cadherin, vimentin and α-SMA by Western blot (n=4). (E) Representative PTEN, PI3K, p-AKT and t-AKT bands by Western blot in HK-2 cells. (F) Densitometric analysis of PTEN, PI3K, p-AKT and t-AKT by Western Blot (n=4). Data are expressed as the mean ± SD. *P < 0.05 vs. the NG group. #P < 0.05 vs. the HG group. NG: normal glucose; MA: mannitol; HG: high glucose; TP: triptolide.

Figure 4

PTEN is a direct target of miR-188-5p and triptolide can promote PTEN expression. (A) Quantification of miR-188-5p gene expression in the HK-2 cells (n=4). (B) Quantification of miR-188-5p gene expression in rat kidneys (n=15). (C) miR-188-5p sequence and its potential binding sites in the wild-type PTEN-3ʹ-UTR. The complementary binding site was replaced in the mutant PTEN 3ʹ-UTR. (D) Dual-luciferase reporter assay with wild-type PTEN-3ʹ-UTR and mutated PTEN- 3ʹ-UTR reporter gene in 293T cells transfected with miR-188-5p mimic or miR-mNC for 24 h (n=4). (E) Quantification of PTEN gene expression in the HK-2 cells (n=4). Data are expressed as the mean ± SD. **P < 0.01 vs. the miR-mNC group. *P < 0.05 vs. the NG group. # P < 0.05 vs. the HG group. NC: normal control; DKD: diabetic kidney disease; TP: triptolide; NG: normal glucose; MA: mannitol; HG: high glucose; miR-188-5pm: miR-188-5p mimic; miR-mNC: negative control of miR-188-5p mimic.

Figure 5

miR-188-5p downregulation alleviated HG-induced EMT in vitro. (A) Representative PTEN, PI3K, p-AKT and t-AKT bands by Western blot in HK-2 cells. (B) Densitometric analysis of PTEN, PI3K, p-AKT and t-AKT by Western blot (n=4). (C) Representative E-cadherin, vimentin and α-SMA bands by Western blot in HK-2 cells. (D) Densitometric analysis of E-cadherin, vimentin and α-SMA by Western blot (n=4). Data are expressed as the mean ± SD. *P < 0.05 vs. the NG group. #P < 0.05 vs. the HG group. NG: normal glucose; MA: mannitol; HG: high glucose; TP: triptolide; miR-188-5pi: miR-188-5p inhibitor; miR-iNC: negative control of miR-188-5p inhibitor.

Figure 6

miR-188-5p overexpression reversed the anti-EMT effects of triptolide in vitro. (A) Representative PTEN, PI3K, p-AKT and t-AKT bands by Western blot in HK-2 cells. (B) Densitometric analysis of PTEN, PI3K, p-AKT and t-AKT by Western blot (n=4). (C) Representative E-cadherin, vimentin and α-SMA bands by Western blot in HK-2 cells. (D) Densitometric analysis of E-cadherin, vimentin and α-SMA bands by Western blot (n=4). Data are expressed as the mean ± SD. *P < 0.05 vs. the HG group. #P < 0.05 vs. the HG+TP group. NG: normal glucose; MA: mannitol; HG: high glucose; TP: triptolide; miR-188-5pm: miR-188-5p mimic; miR-mNC: negative control of miR-188-5p mimic.