Potential Mechanism Of Sulforaphane In The Renoprotective Effects On Diabetic Nephropathy In Mice

Oxidative stress which contributes to the occurrence and development of diabeticnephropathy (DN) is an importantpathogenesis of DN. As a transcription factor,Nuclear factor (erythroid-derived2)-like2(Nrf2) can increase the transcription andexpression of endogenous antioxidatant proteins, such as heme oxygenase1(HO-1),NAD (P) H-quinone oxidoreductase1(NQO1), superoxide dismutase (SOD), catalase(CAT) and Glutamate cysteine ligase (GCL). There were studies showing thatsulforaphane (SFN) can protect DN via up-regulation of Nrf2. However, it has notbeen identified which Nrf2downstream antioxidatant plays the key role in SFNprotection against DN. In addition, evidence showed that metallothionein (MT)expression in rat liver was drastically up-regulated (up to10-fold) after SFN treatment.As a potent endogenous antioxidatant protein, MT can reduce oxidative stress,slowing the progression of DN. However, it has not been addressed whether SFN canprotect DN by increasing kidney MT expression and whether MT is Nrf2downstreamantioxidatant.In order to identify whether SFN can increase renal MT expression, we set up theanimal model of type2diabetes mellitus (T2DM) in C57BL/6J wild type mice byhigh-fat diet and STZ injection. After diabetes onset, mice were treated with SFN for4months. The mice were divided into control group, SFN group, DM group, DM/SFNgroup. After4months of SFN treatment, mice were killed and the blood, the urine and thekidney tissue were collected. The results showed that compared with control group, theurinary albumin to creatinine ratio (UACR), kidney pathological changes, inflammation,fibrosis and oxidative stress were significantly increased accompanied by theup-regulation of HDAC2and HDAC4. SFN treatment for4months significantlyattenuated these changes and reduced HDAC2,4expression in diabetic kidney, theseeffects were accompanied by the up-regulation of Nrf2and MT. Mechanistically, SFNreduced the expression of HDAC2and HDAC4, inducing histone hyperacetylationatMT1promoter. Besides, SFN increased Nrf2expression and Nrf2binding at MT1 promoterin diabetic kidney, resulting in MT transcriptional upregulation.In order to determine whether SFN protect DN entirely depend on Nrf2, and tofurther confirm MT is the Nrf2downstream antioxidant, we set up the animal modelof T2DM in C57BL/6J wild type mice and C57BL/6J Nrf2knockout mice,respectively. After diabetes onset, mice were treated with SFN for4months. BothC57BL/6J wild type mice and C57BL/6J Nrf2knockout mice were divided intocontrol group, SFN group, DM group, DM/SFN group. The results showed that inwild type mice, SFN significantly reduced UACR, renal inflammation and fibrosis ofdiabetic mice, and these effects were accompanied by the up-regulation of Nrf2andMT. In Nrf2knockout mice, SFN completely lost its protection against DN and lostits function to upregulate MT.In order to determine whether MT plays a role in SFN protection against DN, weset up the animal model of T2DM in129S1wild type mice and129S1MT knockoutmice, respectively. After diabetes onset, mice were treated with SFN for4months.Both129S1wild type mice and129S1MT knockout mice were divided into controlgroup, SFN group, DM group, DM/SFN group. The results showed that in wild typemice, SFN reduced UACR of diabetic mice by55%; In MT knockout mice, SFNreduced UACR of diabetic mice by27%, and there was significant difference betweenthese two reductions. In addition, in order to know whether MT influence Nrf2expression, we measured renal Nrf2expression in both129S1wild type mice and129S1MT knockout mice. The results showed that in both wild type mice and MTknockout mice, diabetes reduced Nrf2expression and SFN treatment significantlyincreased Nrf2expression both at mRNA and protein level.The above results showed that SFN can prevent kidney from T2DM-inducedrenal damage, and this renoprotection totally depends Nrf2, partially depends on MT.As one of Nrf2downstream antioxidants, MT plays an important role in SFNprotection against DN. The potential mechanisms by which SFN protected against DNare as follows: SFN reduced the expression of HDAC2and HDAC4in diabetic kidney,inducing histone hyperacetylationat MT promoter. Histone hyperacetylation ledtorelaxed chromatinstructure which provided binding sites for transcription factors tofacilitate MT transcription. Besides, SFN significantly increased Nrf2expression andNrf2binding at MT promoter in diabetic kidney, resulting in MT transcriptionalupregulation. Consequently, SFN reduced diabetes-induced oxidative damage in kidney and alleviated DN.The study’s main points of innovation include:(1) For the first time, we demonstrated that as a HDAC inhibitor and Nrf2activator, SFN protected DN via upregulation of MT in mouse model of T2DM.(2) Although previous studies showed that Nrf2and MT were simultaneouslyinduced or inhibited under a specific condition, the present study, for the first time,demonstrated that MT was Nrf2downstream antioxidant using ChIP assay and Nrf2knockout mice and MT knockout mice.(3) For the first time, the study compared the effects of SFN on129S1wild typediabetic mice and129S1MT knockout diabetic mice, demonstrating the importantrole of MT in SFN protection against DN.(4) For the first time, the study demonstrated the epigenetic mechanisms bywhich HDAC inhibitor upregulated MT, and thus protecting against DN.