Metallothionein plays a prominent role in the prevention of diabetic nephropathy by sulforaphane via up-regulation of Nrf2

Abstract

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 1 diabetes via up-regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, it has not been addressed whether SFN also prevents DN from type 2 diabetes or which Nrf2 downstream gene(s) play(s) the key role in SFN renal protection. Here we investigated whether Nrf2 is required for SFN protection against type 2 diabetes-induced DN and whether metallothionein (MT) is an Nrf2 downstream antioxidant using Nrf2 knockout (Nrf2-null) mice. In addition, MT knockout mice were used to further verify if MT is indispensable for SFN protection against DN. Diabetes-increased albuminuria, renal fibrosis, and inflammation were significantly prevented by SFN, and Nrf2 and MT expression was increased. However, SFN renal protection was completely lost in Nrf2-null diabetic mice, confirming the pivotal role of Nrf2 in SFN protection from type 2 diabetes-induced DN. Moreover, SFN failed to up-regulate MT in the absence of Nrf2, suggesting that MT is an Nrf2 downstream antioxidant. MT deletion resulted in a partial, but significant attenuation of SFN renal protection from type 2 diabetes, demonstrating a partial requirement for MT for SFN renal protection. Therefore, the present study demonstrates for the first time that as an Nrf2 downstream antioxidant, MT plays an important, though partial, role in mediating SFN renal protection from type 2 diabetes.

Keywords: Diabetes; Fibrosis; Inflammation; Kidney; Oxidative stress.

Fig. 1

Effects of SFN on kidney function and pathological changes. A type 2 diabetes model of C57BL/6J mice was induced by a single dose of STZ injection with HFD feeding for 3 months, and then diabetic and age-matched control mice were treated with SFN (0.5 mg/kg) or vehicle daily, five days per week, along with continually feeding HFD for 4 months. Urinary albumin-to-creatinine ratio (UACR, A) and kidney-weight-to-tibia length (B) were determined in all mice. H&E staining was performed to present kidney remodeling (C). Sirius Red staining with semiquantification for renal fibrosis (D). Immunohistochemical (IHC) staining (E) was performed to test localization of renal fibrosis, inflammation, and oxidative damage, using antibodies against TGF-β1, VCAM-1, and 3-NT. Bar = 100 µM. Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. control group (Ctrl); # p < 0.05 vs. diabetic group (DM).

Fig. 2

SFN significantly attenuated diabetes-induced renal fibrosis, inflammation, and oxidative damage. Western blotting was performed to evaluate the expressions of profibrotic mediators [TGF-β1 (A) and CTGF (B)], inflammatory cytokines [PAI-1 (C) and VCAM-1 (D)], and oxidative damage accumulation [3-NT (E) and 4-HNE (F)]. Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. Abbreviations are the same as in Fig. 1.

Fig. 3

SFN induced Nrf2 expression and function along with metallothionein (MT) expression under both diabetic and nondiabetic conditions. Total (A) and nuclear (B) Nrf2 protein levels were measured by western blotting. NQO1 mRNA, which represents Nrf2 function, was determined by real-time PCR (C). Keap1 mRNA (D) and protein (C) levels were evaluated by RT-PCR and western blotting. MT1 mRNA (F) and protein (G), as interest targets of the present study, were also evaluated. Localization of Nrf2 and MT protein induced by SFN was determined by IHC staining (H). Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. Abbreviations are the same as Fig. 1.

Fig. 4

Nrf2 deletion resulted in complete abolition of SFN renal protection from albuminuria, fibrosis, and inflammation. To define the role of Nrf2 in SFN protection from type 2 diabetes-induced DN, Nrf2 knockout (Nrf2-null) and wild-type (WT) C57BL/6J (C57) mice were induced to type 2 diabetes as described in Fig. 1. UACR (A), protein levels of CTGF (B), VCAM-1 (C), 3-NT (D), and 4-HNE (E) for renal function, fibrosis, inflammation, and oxidative stress were evaluated by western blotting in all mice. Diabetes-induced functional and pathological changes (fold) were compared between WT and Nrf2-null mice (F). To further confirm the western blot result for renal fibrosis, morphology of these kidney tissues was examined with PAS staining (G) to evaluate glomerular area (H) and mesangial matrix expansion (I). Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. ‡ p <0.05 vs. C57 (χ² test). Abbreviations are the same as in Fig. 1.

Fig. 5

SFN lost the function of inducing NQO1 and MT expression in the absence of Nrf2. Total Nrf2 protein levels were first evaluated to confirm both the Nrf2-up-regulating effect of SFN in WT mice (A, left panel) and the Nrf2-deletion effect in Nrf2-null mice (A, right panel). NQO1 mRNA (B), MT1 mRNA (C), and MT protein (D) were also evaluated in both WT and Nrf2-null mice. Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. Abbreviations are the same as in Fig. 1.

Fig. 6

MT deletion led to partial abolition of SFN protection against diabetes-induced albuminuria, renal fibrosis, and inflammation. To test whether MT is really required for SFN renal protection, MT-null and their WT 129S1 mice were induced to type 2 diabetes as described in Fig. 1. UACR (A), protein levels of CTGF (B), VCAM-1 (C), 3-NT (D), and 4-HNE (E) were determined by western blotting in all mice. Diabetes-induced functional and pathological changes (fold) between WT and MT-null mice and the decreased percentages of these functional and pathological changes with SFN between WT and MT-null diabetic mice were compared (F). Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. ǂ p< 0.05 vs. 129S1 DM. ‡ p <0.05 vs. WT129 or WT129/DM correspondingly (χ² test) in all mice.

Fig. 7

SFN retained the ability to induce Nrf2 and NQO1 expression in the absence of MT. MT protein level (A) was evaluated to confirm the effect of MT deletion. With the aim of testing if MT deletion has impact on Nrf2 expression and its ability to activate NQO1 transcription, Nrf2 mRNA (B) and nuclear Nrf2 protein (C), as well as NQO1 mRNA (D), were determined. Data are presented as mean ± SD (n = 6 per group). * p < 0.05 vs. ctrl; # p < 0.05 vs. DM. Abbreviations are the same as in Fig. 1.

Fig. 8

Possible mechanism by which SFN ameliorates DN. Type 2 diabetes induces oxidative stress to enhance renal inflammation and fibrosis, which consequently leads to development of DN. Type 2 diabetes decreases Nrf2 and MT expression, resulting in prolonged amplification of oxidative damage. SFN functions through Nrf2 to activate the expression of downstream antioxidant MT and NQO1. MT offers almost half of SFN protection against DN in addition to other Nrf2 downstream antioxidants, such as NQO1. →, activation; ┤, inhibition. Red arrows and question marker for SFN effect on Nrf2 indicate that since we have found increased expression of Nrf2 mRNA by SFN in the present study, it is unclear whether SFN directly up-regulated Nrf2 expression by some uncertain mechanisms alone or in combination with the stabilization of Nrf2 content by oxidizing Keap1.