SP600125 suppresses Keap1 expression and results in NRF2-mediated prevention of diabetic nephropathy

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   Figure 1

   Deletion of the Nrf2 gene led to a complete abolishment of SP600125’s protection against diabetes-induced albuminuria and renal pathological change. Eight-week-old C57BL/6 WT and Nrf2 KO male mice were induced to diabetes by streptozotocin. (A) Blood glucose levels were determined every four weeks post diabetes onset. Urinary albumin and creatinine were recorded at 24 weeks post diabetes onset with (B) UACR calculated. To evaluate renal pathological change, (C) PAS and (D) Masson’s trichrome staining were performed, with (E) glomerular area and (F) mesangial matrix expansion quantified from PAS staining and (G) Masson’s positive area quantified from Masson’s trichrome staining. For (E, F and G), the data are normalized to WT Ctrl. All the data are presented as means ± s.d. (n = 8). *P < 0.05 vs WT Ctrl; ^† P < 0.05 vs KO Ctrl; ^‡ P < 0.05 vs WT DM; ^§ P < 0.05 vs KO DM. Bar = 50 µm. Scatter plots: solid up triangle, WT Ctrl; solid down triangle, WT Ctrl/JNKi; solid left triangle, WT DM; solid right triangle, WT DM/JNKi; hollow up triangle, KO Ctrl; hollow down triangle, KO Ctrl/JNKi; hollow left triangle, KO DM; hollow right triangle, KO DM/JNKi. Bars: white, Ctrl; light gray, Ctrl/JNKi; dark gray, DM; black, DM/JNKi. Ctrl, control; DM, diabetes mellitus; JNKi, the JNK inhibitor SP600125; KO, knockout; PAS, Periodic acid-Schiff; UACR, urinary albumin to creatinine ratio; WT, wild type.
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   Figure 2

   NRF2 was required for SP600125’s alleviation of the diabetes-induced renal oxidative stress, inflammation and fibrosis. To further test the role of NRF2 in SP600125’s protection against diabetes-induced renal injury, IHC staining was performed to assess renal expression of (A) 3-NT, an indicator of nitrosative damage. Further, renal (B) MDA levels were measured by an MDA assay kit and protein levels of (C) iNOS, (D) TNF-α, (E) VCAM-1, (F) TGF-β1 and (G) CTGF were determined by Western blot. For (B), the data is normalized to WT Ctrl. For (C, D, E, F and G), the date is normalized to either WT Ctrl or KO Ctrl, respectively. All the data are presented as means ± s.d. (n = 8). *P < 0.05 vs WT Ctrl; ^† P < 0.05 vs KO Ctrl; ^‡ P < 0.05 vs WT DM; ^§ P < 0.05 vs KO DM. Bars: white, Ctrl; light gray, Ctrl/JNKi; dark gray, DM; black, DM/JNKi. 3-NT, 3-nitrotyrosine; CTGF, connective tissue growth factor; IHC, inmmunohistochemical; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; TGF-β1, transforming growth factor beta 1; TNF-α, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecule 1. Other abbreviations are the same as those in Fig. 1.
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   Figure 3

   SP600125 preserved renal NRF2 protein and facilitated NRF2 nuclear translocation and function, without altering Nrf2 mRNA. To test the effect of SP600125 on Nrf2 gene expression and function, (A) Nrf2 mRNA, protein levels of (B) t-NRF2 and (C) n-NRF2 were determined in both types of the mice. In order to evaluate NRF2 nuclear translocation, (D) ratio of n-NRF2/Histione H3 to t-NRF2/GAPDH was calculated. Further, NRF2 function was evaluated by determining the mRNA expression of (E) Ho1 and (F) Nqo1. IHC staining of (G) HO1 and (H) NQO1 was further performed to assess the status of renal antioxidant activity. The data are normalized to WT Ctrl and presented as means ± s.d. (n = 8). *P < 0.05 vs WT Ctrl; ^† P < 0.05 vs WT DM. Bars: white, Ctrl; light gray, Ctrl/JNKi; dark gray, DM; black, DM/JNKi. Ho1, heme oxygenase 1; n-NRF2, nuclear NRF2; Nqo1, NAD(P)H dehydrogenase (quinone) 1; t-NRF2, total cellular NRF2. Other abbreviations are the same as those in Figs 1 and 2.
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   Figure 4

   SP600125 decreased Keap1 expression through inhibition of JNK activity. With the aim of exploring the mechanism by which SP600125 activates NRF2, Keap1 (A) mRNA and (B) protein, as well as ratios of (C) p-JNK to t-JNK and (D) p-c-Jun to t-c-jun were determined in the kidneys of the WT and Nrf2 KO mice. To further define the effect of JNK inhibition on Keap1 expression, MMCs were treated with HG, in the presence of either SP600125 or JNK siRNA, with ratios of (E) p-JNK to t-JNK, t-JNK to GAPDH, p-JNK to GAPDH and (F) p-c-Jun to t-c-jun, as well as Keap1 (G) mRNA and (H) protein determined. For (A, B, C and D), the data is normalized to respective Ctrls and presented as means ± s.d. (n = 8). *P < 0.05 vs WT Ctrl; ^† P < 0.05 vs KO Ctrl; ^‡ P < 0.05 vs WT DM; ^§ P < 0.05 vs KO DM. Bars: white, Ctrl; light gray, Ctrl/JNKi; dark gray, DM; black, DM/JNKi. For (E, F, G and H), the data are normalized to HG and presented as means ± s.d. (n = 3). *P < 0.05 vs HG; ^† P < 0.05 vs HG/NC. Bars: white, HG; light gray, HG/JNKi; dark gray, HG/RFect^PM; black, HG/NC; white with stripes, HG/siJNK. HG, high glucose; Keap1, Kelch-like ECH-associated protein 1; NC, negative control siRNA; p-c-Jun, phosphorylated c-Jun; p-JNK, phosphorylated JNK; RFect^PM, the transfection reagent; siJNK, JNK siRNA; t-c-Jun, total c-Jun; t-JNK, total JNK. Other abbreviations are the same as those in Fig. 1.
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   Figure 5

   Both SP600125 and JNK siRNA preserved NRF2 protein and enhanced its nuclear translocation and function. In order to test the effect of JNK inhibition on NRF2 expression and function, MMCs were subjected to HG, in the presence of either SP600125 or JNK siRNA. (A) t-NRF2 and (B) n-NRF2 levels were determined by Western blot. (C) n-NRF2/Histione H3 to t-NRF2/GAPDH was calculated to reflect NRF2 nuclear translocation. mRNA levels of (D) Ho1 and (E) Nqo1 were determined by RT-PCR. The data are normalized to HG and presented as means ± s.d. (n = 3). *P < 0.05 vs HG; ^† P < 0.05 vs HG/NC. Bars: white, HG; light gray, HG/JNKi; dark gray, HG/RFect^PM; black, HG/NC; white with stripes, HG/siJNK; Abbreviations are the same as those in Figs 3 and 4.
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   Figure 6

   JNK inhibition by either SP600125 or JNK siRNA decreased oxidative damage and expression of inflammatory and fibrotic genes in HG-treated MMCs. SP600125 and JNK siRNA were further tested for their roles in alleviating HG-induced oxidative stress, inflammation and fibrosis in MMCs. (A) MDA levels and mRNA expression of (B) iNos, (C) Vcam-1, (D) Icam-1, (E) Fn and (F) Col4 were determined. The data are normalized to HG and presented as means ± s.d. (n = 3). *P < 0.05 vs HG; ^† P < 0.05 vs HG/NC. Bars: white, HG; light gray, HG/JNKi; dark gray, HG/RFect^PM; black, HG/NC; white with stripes, HG/siJNK. Col4, collagen 4; Fn, fibronectin. Other abbreviations are the same as those in Figs 2 and 4.

• © 2018 Society for Endocrinology