Sulforaphane prevents type 2 diabetes-induced nephropathy via AMPK-mediated activation of lipid metabolic pathways and Nrf2 anti-oxidative function.

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 2 diabetes (T2D) by up-regulating nuclear factor (erythroid-derived 2)-like 2 (Nrf2). AMP-activated protein kinase (AMPK) can attenuate the pathogenesis of DN by improving renal lipotoxicity along with the activation of Nrf2-mediated anti-oxidative signaling. Therefore, we investigated whether AMPKα2, the central subunit of AMPK in energy metabolism, is required for SFN protection against DN in T2D, and whether potential crosstalk occurs between AMPKα2 and Nrf2. AMPKα2 knockout (Ampkα2-/-) mice and wild-type mice were fed a high-fat diet (HFD) or a normal diet (ND) to induce insulin resistance, followed by streptozotocin injection to induce hyperglycemia, as a T2D model. Both T2D and control mice were treated with SFN or vehicle for three months. At the end of the three-month treatment, all mice were maintained only on HFD or ND for an additional three months without SFN treatment. Mice were sacrificed at 6th month after T2D onset. Twenty-four-hour urine albumin at 3rd and 6th months was significantly increased as renal dysfunction, along with significant renal pathological changes and biochemical changes including renal hypertrophy, oxidative damage, inflammation, and fibrosis in wild-type T2D mice, which were prevented by SFN in certain extends, but not in Ampkα2-/- T2D mice. SFN prevention of T2D-induced renal lipotoxicity was associated with AMPK-mediated activation of lipid metabolism and Nrf2-dependent anti-oxidative function in wild-type mice, but not in SFN-treated Ampkα2-/- mice. Therefore, SFN prevention of DN is AMPKα2-mediated activation of probably both lipid metabolism and Nrf2 via AMPK/AKT/GSK3β/Fyn pathways.


Introduction
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide [1]. 45 Although hyperglycemia and insulin resistance are generally considered to be the primary causative 46 factors for DN, mounting evidence suggests that lipid accumulation is a crucial contributor to DN, 47 especially in type 2 diabetes (T2D) [2]. Renal lipid accumulation in the patients with T2D, which is 48 caused by increased lipid uptake and decreased lipid oxidation, is associated with renal inflammation, . In addition, SFN was also found to inhibit adipogenesis accompanied 58 with activation of AMP-activated protein kinase (AMPK) in recent study [6], suggesting that SFN 59 could play an important role in improving dyslipidemia probably through AMPK signaling. These 60 studies indicated that SFN may alleviates diabetic complications through multiple mechanisms, 61 including Nrf2-dependent and -independent pathways, and noticed us a potential role for interactions 62 between SFN and AMPK-mediated lipid metabolism. Therefore, we sought to determine whether SFN 63 could activate AMPK-mediated lipid metabolism pathways to protect against kidney damage in T2D 64 in the current work, which is as yet unknown. SFN dosage in the present study was based on our previous studies [4,16] and other studies [17,18]. 119 In addition, there were two previous studies [19,20] where SFN was given mice either by oral at the 120 dose of 110 µmol/kg [19] or intraperitoneally at the dose of about 63.8 mg/kg [20] and their plasma 121 peak concentrations of SFN were 6.66 µM at 2 hr or 210 µM at 1 hr, respectively. Based on these two 122 studies, we estimated that the serum peak concentration of the SFN used here should be 0.17-1.65 123 µM. Since we treated diabetic mice for three months, the purpose of SFN treatment is to preserve the   previous studies [4, 16], we did not find any significant side effects of SFN on non-diabetic kidneys.

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In addition, SFN effect on nondiabetic kidney is included in WT mice, therefore, KO mice were only  During the 6-month experimental period, tail vein blood glucose was measured for five times to

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After three months of HFD or ND feeding, an intraperitoneal glucose tolerance testing (IPGTT) 143 was performed. After 6h of fasting, we measured tail vein blood glucose, which served as the 0-min 144 time point. Glucose (1.5 mg/g body weight) was then injected IP, followed by 15, 30, 60 and 120-min 145 tail vein blood glucose measurements. The area under the curve (AUC) using the trapezoid rule was 146 calculated to determine glucose tolerance.  1:100 dilution), transforming growth factor β1 (TGF-β1, 1:100 dilution), and collagen-1 (COL-1, 157 1:100 dilution) (all from Abcam, Cambridge, MA) were used for IHC.        Furthermore, all these changes were slightly severe in Ampkα2 -/mice compared to that in WT mice in 230 term of the fold changes between two strain diabetic mice relative to controls ( Figure 2D), suggesting 231 that AMPK deficiency may potentially exacerbate T2D-induced glomerular hypertrophy and increased 232 mesangial matrix expansion. The renal fibrosis, which was defined by increased collagen 233 accumulation, was analyzed by Sirius Red staining. The result showed that renal collagen disposition 234 was markedly increased both in diabetic WT and Ampkα2 -/mice, but SFN treatment reduced 235 diabetes-induced collagen accumulation only in diabetic WT mice. However, although collagen 236 deposition in the SFN-treated group was significantly lower than that in the T2D group, it was still 237 notably higher than Ctrl group. Therefore, SFN cannot completely prevent diabetes-induced renal 238 fibrosis, but ameliorate it ( Figure 2E, F).

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To explore the fibrotic pathways, the protein expression of TGF-β1 a major fibrotic initiator 240 was examined by Western blot ( Figure 3A), which was significantly increased in both WT and Ampkα2 -/diabetic groups and that was preventable only in WT, but not in Ampkα2 -/-, diabetic mice.

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To further define the fibrotic response, FN as one of fibrotic end-products was examined for its 243 expression ( Figure 3B) and renal structural location ( Figure 3C) was examined by Western blot and 244 immunohistochemical stain and showed the significant increase in both WT and Ampkα2 -/diabetic 245 groups, which was preventable only in WT, but not in Ampkα2 -/-, diabetic mice. Similarly, another 246 fibrotic end-product Col-1 was also showed significant increase in T2D kidney and its prevention by 247 SFN ( Figure 3D). Importantly FN mainly accumulates in glomeruli while COL-1 mainly accumulates 248 in renal interstitium ( Figure 3C, D).  improved diabetes-increased these protein levels in WT mice, but not in Ampkα2 -/mice. Considering 258 protein nitration is caused by peroxynitrite formation, which is derived from the interaction of NO 259 with superoxide, we also revealed the increased level of nitric oxide in T2D group, an effect that can 260 be improved by SFN in WT mice, but not in Ampkα2 -/mice ( Figure 4E).

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To explore the potential mechanism for AMPK-mediated induction of Nrf2 activation in diabetic   (Figures 1-4).  Subsequently, the activated AKT inactivated its downstream effector protein GSK3β by 359 phosphorylating it at Ser9 residue ( Figure 8A, B), which was also demonstrated by another study [14].    Competing interests 456 The authors declare that they have no competing interests.     Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191088