Diabetes Metab J.  2021 Nov;45(6):909-920. 10.4093/dmj.2020.0168.

Sulforaphane Ameliorates Diabetes-Induced Renal Fibrosis through Epigenetic Up-Regulation of BMP-7

• Kong L
• Wang H
• Li C
• Cheng H
• Cui Y
• Liu L
• Zhao Y

• KMID: 2522730
• DOI: http://doi.org/10.4093/dmj.2020.0168

Abstract

Background
The dietary agent sulforaphane (SFN) has been reported to reduce diabetes-induced renal fibrosis, as well as inhibit histone deacetylase (HDAC) activity. Bone morphologic protein 7 (BMP-7) has been shown to reduce renal fibrosis induced by transforming growth factor-beta1. The aim of this study was to investigate the epigenetic effect of SFN on BMP-7 expression in diabetes-induced renal fibrosis.
Methods
Streptozotocin (STZ)-induced diabetic mice and age-matched controls were subcutaneously injected with SFN or vehicle for 4 months to measure the in vivo effects of SFN on the kidneys. The human renal proximal tubular (HK11) cell line was used to mimic diabetic conditions in vitro. HK11 cells were transfected to over-express HDAC2 and treated with high glucose/palmitate (HG/Pal) to explore the epigenetic modulation of BMP-7 in SFN-mediated protection against HG/Pal-induced renal fibrosis.
Results
SFN significantly attenuated diabetes-induced renal fibrosis in vivo. Among all of the HDACs we detected, HDAC2 activity was markedly elevated in the STZ-induced diabetic kidneys and HG/Pal-treated HK11 cells. SFN inhibited the diabetes-induced increase in HDAC2 activity which was associated with histone acetylation and transcriptional activation of the BMP-7 promoter. HDAC2 over-expression reduced BMP-7 expression and abolished the SFN-mediated protection against HG/Pal-induced fibrosis in vitro.
Conclusion
Our study demonstrates that the HDAC inhibitor SFN protects against diabetes-induced renal fibrosis through epigenetic up-regulation of BMP-7.

Keyword

Bone morphogenetic protein 7; Diabetic nephropathies; Histone deacetylase 2; Sulforafan

Figure

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  Fig. 1. Effects of sulforaphane (SFN) on diabetes-induced systemic changes and renal disfunction. (A) Urinary albumin to creatinine ratio (UACR), (B) kidney weight/tibia length, (C) blood glucose, and (D) body weight were measured in all mice. Data are presented as mean±standard deviation (n=at least 6 per group). DM, diabetes mellitus. aP<0.05 vs. control, bP<0.05 vs. DM group.

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  Fig. 2. Sulforaphane (SFN) alleviates diabetes-induced glycogen and collagen accumulation in the kidney. Kidney pathology was examined with Periodic acid Schiff (PAS; A, ×400) and Masson’s trichrome staining (B, ×400) in all mice. Mesangial matrix expansion (C) was quantified from PAS staining and fibrotic accumulation (D) was quantified from Masson’s trichrome staining. Glycogen- and collagen-positive areas are displayed in microscopic images of PAS and Masson’s trichrome staining, respectively. Data are presented as mean±standard deviation (n=at least 6 per group). DM, diabetes mellitus. aP<0.05 vs. control, bP<0.05 vs. DM group.

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  Fig. 3. Sulforaphane (SFN) attenuated diabetes-induced renal fibrosis. Protein expression of collagen I (A), collagen IV (B), fibronectin (FN) (C), and α-smooth muscle actin (α-SMA) (D) were detected using Western blot analysis. Data are presented as mean±standard deviation (n=at least 6 per group). DM, diabetes mellitus. aP<0.05 vs. control, bP<0.05 vs. DM group.

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  Fig. 4. Sulforaphane (SFN)-induced histone acetylation is involved in the up-regulation of bone morphologic protein 7 (BMP-7). BMP-7 mRNA levels (A) and protein expression (B) were detected using real-time polymerase chain reaction and Western blot analysis, respectively. Expression of H3K9/14Ac was assessed using Western blot analysis (C). H3K9/14Ac levels in the BMP-7 promoter were measured using the chromatin immunoprecipitation (ChIP) assay (D). Data are presented as mean±standard deviation (n=at least 6 per group). DM, diabetes mellitus; IgG, immunoglobulin G. aP<0.05 vs. control, bP<0.05 vs. DM group.

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  Fig. 5. Histone deacetylase 2 (HDAC2) is a crucial regulator of diabetes-induced renal fibrosis. (A) Effect of streptozotocin-induced diabetes on HDAC activity (n=at least 6 per group). (B) Effect of hyperglycemia/hyperlipidemia on HDAC activity in human renal proximal tubular (HK11) cells. (C, D) HK11 cells were transfected with an HDAC2 over-expression plasmid. The transfection efficiency was determined using Western blot analysis (C). Effects of HDAC2 over-expression on bone morphologic protein 7 (BMP-7) expression (C) and hyperglycemia/hyperlipidemia-induced renal fibrosis (D) were determined using Western blot analysis. Data are presented as mean±standard deviation of four experiments. DM, diabetes mellitus; HG/Pal, high glucose/palmitate; NC, negative control transfected with empty vector; FN, fibronectin; α-SMA, α-smooth muscle actin. aP<0.05 vs. control or NC correspondingly, bP<0.05 vs. NC/HG/Pal.

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  Fig. 6. Sulforaphane (SFN) activates bone morphologic protein 7 (BMP-7)-Smad1/5/8 and suppresses transforming growth factor beta1 (TGF-β1)-Smad2/3 pathways by inhibiting histone deacetylase 2 (HDAC2) activity. (A) Effect of SFN on HDAC2 activity. (B) Effect of SFN on the expression of phospho-Smad2/3 and TGF-β1. (C) Effect of SFN on the expression of phospho-Smad1/5/8. Data are presented as mean±standard deviation (n=at least 6 per group). (D, E, F, G) Effects of HDAC2 over-expression on SFN-mediated prevention of hyperglycemia/hyperlipidemia-induced fibrosis and on SFN-induced BMP-7 expression. Data are presented as mean±standard deviation of four experiments. DM, diabetes mellitus; HG/Pal, high glucose/palmitate; FN, fibronectin; NC, negative control transfected with empty vector. aP<0.05 vs. control, bP<0.05 vs. DM group, cP<0.05 vs. NC/control or HDAC2/control correspondingly, dP<0.05 vs. NC/HG/Pal.

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