Oridonin ameliorates renal fibrosis in diabetic nephropathy by inhibiting the Wnt/β-catenin signaling pathway

Abstract

Diabetic nephropathy (DN) is one of the most serious and frequent complications among diabetes patients and presently constitutes vast the cases of end-stage renal disease worldwide. Tubulointerstitial fibrosis is a crucial factor related to the occurrence and progression of DN. Oridonin (Ori) is a diterpenoid derived from rubescens that has diverse pharmacological properties. Our previous study showed that Ori can protect against DN by decreasing the inflammatory response. However, whether Ori can alleviate renal fibrosis in DN remains unknown. Here, we investigated the mechanism through which Ori affects the Wnt/β-catenin signaling pathway in diabetic rats and human proximal tubular epithelial cells (HK-2) exposed to high glucose (HG) levels. Our results revealed that Ori treatment markedly decreased urinary protein excretion levels, improved renal function and alleviated renal fibrosis in diabetic rats. In vitro, HG treatment increased the migration of HK-2 cells while reducing their viability and proliferation rate, and treatment with Ori reversed these changes. Additionally, the knockdown of β-catenin arrested cell migration and reduced the expression levels of Wnt/β-catenin signaling-related molecules (Wnt4, p-GSK3β and β-catenin) and fibrosis-related molecules (α-smooth muscle actin, collagen I and fibronectin), and Ori treatment exerted an effect similar to that observed after the knockdown of β-catenin. Furthermore, the combination of Ori treatment and β-catenin downregulation exerted more pronounced biological effects than treatment alone. These findings may provide the first line of evidence showing that Ori alleviates fibrosis in DN by inhibiting the Wnt/β-catenin signaling pathway and thereby reveal a novel therapeutic avenue for treating tubulointerstitial fibrosis.

Keywords: Oridonin; Wnt/β-catenin; diabetic nephropathy; renal fibrosis.

Figure 1.

Chemical structure of oridonin.

Figure 2.

Effects of Ori on general changes and biochemical indices in diabetic rats. (A) Effect of Ori on blood glucose. (B–D) Effect of Ori on BUN, Scr and 24-h urinary protein concentrations. **p < 0.01 vs. the NC group; ***p < 0.001 vs. the NC group; ^###p < 0.001 vs. the DN group. Ori: oridonin; BUN: blood urea nitrogen; Scr: serum creatinine; NC: normal control; DN: diabetic nephropathy; DN + Ori: diabetic nephropathy + oridonin.

Figure 3.

Effects of Ori on renal pathological changes in diabetic rats. PAS and MT staining of kidney tissues. **p < 0.01 vs. the NC group, ^###p < 0.001 vs. the DN group. PAS: periodic acid-Schiff; MT: Masson’s trichrome; NC: normal control; DN: diabetic nephropathy; DN + Ori: diabetic nephropathy + oridonin.

Figure 4.

Effects of Ori on renal fibrosis in diabetic rats. Immunohistochemical analysis of Col I and FN expression in kidney tissues. ***p < 0.001 vs. the NC group, ^###p < 0.001 vs. the DN group. Col I, collagen I; FN: fibronectin; NC: normal control; DN: diabetic nephropathy; DN + Ori: diabetic nephropathy + oridonin.

Figure 5.

Expression of the Wnt/β-catenin signaling pathway in the kidneys of diabetic rats. (A) Protein expression levels of Wnt4, GSK3β, p-GSK3β, β-catenin and α-SMA in rat kidney tissues. (B) qPCR analysis of the mRNA expression of Wnt4, GSK3β, β-catenin and α-SMA in rat kidney tissues. **p < 0.01 vs. the NC group; ^#p < 0.05 vs. the DN group; ^##p < 0.01 vs. the DN group; ^###p < 0.001 vs. the DN group. p-GSK3β: phospho-glycogen synthase kinase-3β; α-SMA: α-smooth muscle actin; qPCR: quantitative polymerase chain reaction.

Figure 6.

Effects of Ori on cell viability. (A) HK-2 cells were treated with different concentrations of HG (0, 10, 20, or 30 mM) for 48 h, after which their viability was detected via CCK-8 assays. (B) HK-2 cells were treated with HG (30 mM) for different durations (0, 12, 24, and 48 h), and their viability was detected by CCK-8 assays. (C) HK-2 cells were pretreated with different concentrations of Ori (2.5, 5, or 10 μM) for 2 h and then treated with 30 mM HG for 48 h, after which their viability was detected via CCK-8 assays. **p < 0.01 vs. the NG group; ***p < 0.001 vs. the NG group; ^#p < 0.05 vs. the HG group; ^##p < 0.01 vs. the HG group; HG: high glucose; CCK-8: Cell Counting Kit-8; NG: normal glucose control (5.5 mmol/L glucose); MG: mannitol control (19.5 mmol/L mannitol + 5.5 mmol/L glucose); NG + Ori: 5.5 mmol/L glucose + 5 μmol/L oridonin; HG: 30.0 mmol/L glucose; HG + Ori: 30.0 mmol/L glucose + 5 μmol/L oridonin.

Figure 7.

Ori inhibited fibrosis in HG-treated HK-2 cells. (A) Transwell assays were used for the assessment of cell migration. (B & C) Col I and FN were detected by specific immunofluorescence staining. (D) Quantification of the immunofluorescence staining of Col I and FN. **p < 0.01 vs. the NG group; ^#p < 0.05 vs. the HG group; ^##p < 0.01 vs. the HG group; Col I: collagen I; FN: fibronectin; HG: high glucose; NG: normal glucose control (5.5 mmol/L glucose); MG: mannitol control (19.5 mmol/L mannitol + 5.5 mmol/L glucose); NG + Ori: 5.5 mmol/L glucose + 5 μmol/L oridonin; HG: 30.0 mmol/L glucose; HG + Ori: 30.0 mmol/L glucose + 5 μmol/L oridonin.

Figure 8.

Expression of proteins in the Wnt/β-catenin signaling pathway in HG-treated HK-2 cells. (A) Protein expression levels of Wnt4, GSK3β, p-GSK3β, β-catenin and α-SMA in HK-2 cells. (B) qPCR analysis of the mRNA expression of Wnt4, GSK3β, β-catenin and α-SMA in HK-2 cells. **p < 0.01 vs. the NC or NG group, ***p < 0.001 vs. the NC or NG group, ^#p < 0.05 vs. the DN or HG group, ^##p < 0.01 vs. the DN or HG group, and ^###p < 0.001 vs. the DN or HG group. p-GSK3β, phospho-glycogen synthase kinase-3β; α-SMA: α-smooth muscle actin; qPCR: quantitative polymerase chain reaction.

Figure 9.

Ori attenuated renal fibrosis by inhibiting the Wnt/β-catenin signaling pathway. (A) Transwell assays were used for the assessment of cell migration. (B & C) Detection of Col I and FN expression via immunofluorescence staining. (D) Quantification of the immunofluorescence staining of Col I and FN. (E) Protein expression levels of Wnt4, GSK3β, p-GSK3β, β-catenin and α-SMA in HK-2 cells. (F) qPCR analysis of the mRNA expression of Wnt4, GSK3β, β-catenin and α-SMA in HK-2 cells. The results are representative of three independent experiments. ^#p < 0.05 vs. the HG group, ^##p < 0.01 vs. the HG group, ^###p < 0.001 vs. the HG group. KD: knockdown; OE: overexpression.

Figure 9.

Ori attenuated renal fibrosis by inhibiting the Wnt/β-catenin signaling pathway. (A) Transwell assays were used for the assessment of cell migration. (B & C) Detection of Col I and FN expression via immunofluorescence staining. (D) Quantification of the immunofluorescence staining of Col I and FN. (E) Protein expression levels of Wnt4, GSK3β, p-GSK3β, β-catenin and α-SMA in HK-2 cells. (F) qPCR analysis of the mRNA expression of Wnt4, GSK3β, β-catenin and α-SMA in HK-2 cells. The results are representative of three independent experiments. ^#p < 0.05 vs. the HG group, ^##p < 0.01 vs. the HG group, ^###p < 0.001 vs. the HG group. KD: knockdown; OE: overexpression.