Protective Effects of Berberine on Renal Injury in Streptozotocin (STZ)-Induced Diabetic Mice

Abstract

Diabetic nephropathy (DN) is a serious diabetic complication with renal hypertrophy and expansion of extracellular matrices in renal fibrosis. Epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells may be involved in the main mechanism. Berberine (BBR) has been shown to have antifibrotic effects in liver, kidney and lung. However, the mechanism of cytoprotective effects of BBR in DN is still unclear. In this study, we investigated the curative effects of BBR on tubulointerstitial fibrosis in streptozotocin (STZ)-induced diabetic mice and the high glucose (HG)-induced EMT in NRK 52E cells. We found that BBR treatment attenuated renal fibrosis by activating the nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway in the diabetic kidneys. Further revealed that BBR abrogated HG-induced EMT and oxidative stress in relation not only with the activation of Nrf2 and two Nrf2-targeted antioxidative genes (NQO-1 and HO-1), but also with the suppressing the activation of TGF-β/Smad signaling pathway. Importantly, knockdown Nrf2 with siRNA not only abolished the BBR-induced expression of HO-1 and NQO-1 but also removed the inhibitory effect of BBR on HG-induced activation of TGF-β/Smad signaling as well as the anti-fibrosis effects. The data from present study suggest that BBR can ameliorate tubulointerstitial fibrosis in DN by activating Nrf2 pathway and inhibiting TGF-β/Smad/EMT signaling activity.

Keywords: EMT; Nrf2 pathway; TGF-β/Smad signaling pathway; berberbine; diabetic nephropathy; renal tubular epithelial cells.

Figure 1

Double immunofluorescence staining shows localization of α-SMA (red) and the tubular cell marker lectin (green) in control (a1–3) and diabetic kidneys (b1–3); BBR-treated diabetic kidneys (c1–3) (respectively. Scale bar, 20 μm).

Figure 2

Double immunofluorescence staining shows localization of collagen-I (red) and the tubular cell marker lectin (green) in control (a1–3) and diabetic kidneys (b1–3); BBR-treated diabetic kidneys (c1–3) (respectively. Scale bar, 20 μm).

Figure 3

Antifibrotic effects of BBR were associated with the inhibition of EMT in diabetic kidneys. Representative western blots of α-SMA and collagen-I in the kidneys of control mice, diabetic mice or BBR-treated diabetic mice. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p < 0.01 vs. control group; ## p < 0.01 vs. control diabetic group. n = 8).

Figure 4

BBR enhanced activation of Nrf2/HO-1 signaling in diabetic kidneys. Representative western blots demonstrated that BBR treatment increased protein expression of Nrf2, NQO1 and HO-1 in the kidneys of diabetic mice (n = 8). Each value represents mean ± SEM. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p < 0.01 vs. control group; ## p < 0.01 vs. control diabetic group. n = 8).

Figure 5

Effects of BBR on HG-induced EMT in the NRK-52E cells. (a) The NRK-52E cells were treated with various doses of BBR (3, 30 or 300 μM) for 6, 12, 24, 48 or 72 h, and the cell viability was analyzed by MTT assay. The data were mean ± SEM (n = 5); (b,c) The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of 30 μM BBR. Representative western blot analysis shows that BBR treatment prevents high glucose (HG)-induced (b) E-ca downregulation, as well as (c) á-smooth muscle actin (á-SMA) upregulation at 48 h (n = 6). Values represent mean ± SEM of three independent experiments performed (** p < 0.01 vs. control; * p < 0.05 vs. control; ## p < 0.01 vs. HG; # p < 0.05 vs. HG); (d–j) Phase contrast microscopy shows that BBR treatment prevents non-epitheliod phenotype acquisition of the NRK-52E cells. Scale bar, 20 μm. For confocal microscopy of immunofluorescence-stained samples, cells were fixed and stained with a primary antibody against E-ca (d through g), α-SMA (h through j).Cultures were untreated (d,h) or exposed to HG (e,i) or with the addition of BBR (g,j). Results are representative of two experiments.

Figure 6

Influence of BBR on HG-induced Nrf2/HO-1 signaling in NRK-52E cells. The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of BBR. The expression NQO1, HO-1 and Nrf2 was analyzed using western blot (n = 6). (a–e) The results were representatives of three independent experiments. β-actin was used as loading control. (** p < 0.01 vs. control; ## p < 0.01 vs. HG).

Figure 7

siRNA knockdown of Nrf2 abrogates BBR-induced NQO1 and HO-1 expression. (a) NRK-52E cells were transfected with Nrf2-siRNA and western blot analysis was performed with an antibody against Nrf2 was performed at various time-points following transfection (24, 48 and 72 h). Relative Nrf2 expression levels were calculated and normalized to the loading control. Corresponding protein levels were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM (n = 6; ** p < 0.01, vs. control); (b,c) The cells were divided into six groups as description in this paper. Western blotting was performed with an antibody against NQO1 and HO-1. Relative NQO1 and HO-1 expression levels were calculated and normalized to the loading control. Corresponding protein levels were assessed using densitometry and were expressed in relative intensities. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM (n = 6) (** p < 0.01 vs. control; ## p < 0.01 vs. HG; # p < 0.05 vs. HG; ΔΔ p < 0.01 vs. HG + BBR).

Figure 8

Nrf2 is involved in the inhibitory effect of BBR on the TGF-β/Smad signaling pathway. NRK-52E cells were divided into six groups as mentioned above, corresponding protein levels of Smad2/3, phospho-Smad2/3 and EMT markers were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM (n = 6) (* p < 0.05 vs. control; ** p < 0.01 vs. control; ## p< 0.01 vs. HG; # p < 0.05 vs. HG; ΔΔ p< 0.01 vs. HG + BBR).