Derivative of Bardoxolone Methyl, dh404, in an Inverse Dose-Dependent Manner Lessens Diabetes-Associated Atherosclerosis and Improves Diabetic Kidney Disease
Article Figures & Tables
Figures
- Figure 1
Diabetes-associated lesion formation in the aorta and aortic sinus is attenuated by dh404 after 18 weeks of treatment. Aortas were stained with Sudan IV Herxheimer’s solution, and plaques were stained red (A). Treatment with dh404 at 3 and 10 mg/kg/day resulted in an attenuation of total plaque formation in the aortas of diabetic mice (B), and similar effects were found in the aortic arch (C), thoracic (D), and abdominal regions (E). F: Cryosections of aortic sinus were stained with Oil Red O to detect plaque formation after 18 weeks of dh404 treatment. G: At 3 and 10 mg/kg/day, plaque formation was significantly attenuated in diabetic mice when compared with their vehicle-treated counterparts. Data are mean ± SEM. **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05, ##P < 0.01 vs. D+SO; ^P < 0.05, ^^P < 0.01 vs. as indicated. Abd, abdominal region; Arch, aortic arch; D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; ND, nondiabetic mice; Thor, thoracic.
- Figure 2
Adhesion and inflammatory markers in aorta are reduced by dh404. The gene expression of adhesion and inflammatory markers in the aorta was assessed by qRT-PCR after 5 weeks of treatment (A–F). VCAM-1 protein expression was examined using immunohistochemistry in aortic plaque (G), and the quantitation is shown in H. Data are mean ± SEM. For qRT-PCR analysis, n = 6–10 (MCP-1), 7–10 (TNF-α), and 8–10 (CD36, ICAM-1, p65, and VCAM-1) aortas/group. *P < 0.05, **P < 0.01 vs. ND+SO; #P < 0.05, ##P < 0.01 vs. D+SO. A.U., arbitrary units; D, diabetic mice dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; ND, nondiabetic mice.
- Figure 3
Diabetes-associated oxidative stress is attenuated by dh404 after 18 weeks of treatment. Oxidative stress was assessed by urine levels of 8-isoprostane (A) and 8-OHdG (B) and plasma levels of dROMs (C). All of these markers were attenuated in the diabetic mice by dh404. Nitrotyrosine immunostaining revealed that diabetes was associated with an increase in nitrotyrosine protein expression, and this was attenuated by dh404 at all doses (D and E). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D+SO. D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; ND, nondiabetic mice; U.Carr, Carratelli units.
- Figure 4
dh404 increases antioxidant as well as inflammatory gene expression after 5 weeks of treatment, improves kidney function, and attenuates glomerulosclerosis and tubulointerstitial injury after 18 weeks of treatment. The gene expression of antioxidants NQO1, GSH-S transferase, and GPx1 was increased in the kidney cortex by dh404 after 5 weeks of treatment (A). However, inflammatory genes such as MCP-1 and NF-κB were also upregulated by dh404 (B). Diabetes-associated increase in urinary albumin-to-creatinine ratio (UACR) was reduced by dh404 (C); however, dh404 treatment had no effect on plasma cystatin C levels (D). Glomerulosclerosis was assessed by measuring PAS-stained area per glomerulus (indicative of mesangial expansion) and also scored for GSI. Photomicrographs of representative glomeruli are shown in E. Treatment of dh404 at 3 mg/kg/day for 18 weeks significantly attenuated mesangial expansion (F) and GSI (G). The percentage of tubulointerstitial injury was determined from PAS-stained sections by point-counting and is shown in H. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. ND+SO; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D+SO. A.U., arbitrary units; D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; gcs, glomerular cross-sectional area; ND, nondiabetic mice.
- Figure 5
In vitro analysis of Nrf2-responsive antioxidant genes in NRK cells after dh404 treatment. NRK cells were treated with DMSO, dh404, TGF-β+DMSO, or TGF-β+dh404 for 72 h as detailed in the Research Design and Methods. Gene-expression profiles of HO-1, NQO1, GSH-S transferase, GPx1, GPx2, and GPx3 are shown in A. dh404, in most cases, caused dose-dependent increases in the gene expression of most antioxidants investigated either in the presence or absence of TGF-β treatment. Protein levels for HO-1 and GPx1 after treatment of NRK cells with 0.5 µmol dh404 for 72 h are shown in B. Data are mean ± SEM from five separate experiments for quantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, data were normalized to controls (given arbitrary value of 1) as fold change and then averaged over replicate five experiments. Lane 1, DMSO; lane 2, dh404; lane 3, TGF-β+DMSO; lane 4, TGF-β+dh404. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. CTL+DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. TGF-β+DMSO; ^P < 0.05 CTL+dh404 vs. TGF-β+dh404. A.U., arbitrary units; CTL, control.
- Figure 6
In vitro analysis of Nrf2-responsive anti-inflammatory and fibrosis genes in NRK cells after dh404 treatment. NRK cells were treated with either DMSO, dh404, TGF-β+DMSO, or TGF-β+dh404 for 72 h as detailed in the Research Design and Methods. Gene expression profiles of IL-6, MCP-1, and the p65 subunit of NF-κB are shown in A. Gene expression of fibronectin (FN), collagen I (Col I), and collagen IV (Col IV) are shown in B. Despite decreases in IL-6 expression with increasing doses of dh404, MCP-1 showed a significant increase in gene expression at higher doses of dh404. Collagen I and IV as well as FN gene expression were significantly attenuated by dh404. FN protein levels, shown in C, were significantly reduced after 0.5 µmol dh404 treatment. Data are mean ± SEM from five separate experiments for quantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, data were normalized to control subjects (given arbitrary value of 1) as fold change and then averaged over replicate five experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. CTL+DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. TGF-β+DMSO; ^^^P < 0.001 CTL+dh404 vs. TGF-β+dh404. Lane 1, DMSO; lane 2, dh404; lane 3, TGF-β+DMSO; lane 4, TGF-β+dh404. A.U., arbitrary units; CTL, control.
Tables
- Table 1
Basic characteristics of nondiabetic and diabetic ApoE−/− mice treated with vehicle (SO) or dh404 at conclusion of 18-week study
ND+SO (n = 9) ND+dh-20 (n = 8) D+SO (n = 5) D+dh-3 (n = 7) D+dh-10 (n = 8) D+dh-20 (n = 9) BW, g 30.7 ± 0.9 31.1 ± 0.6 21.8 ± 0.4*** 27.5 ± 0.7**,### 26.8 ± 0.6 ***,### 23.0 ± 0.6*** Right kidney weight/BW, mg/g 6.8 ± 0.2 6.4 ± 0.3 11.1 ± 0.4*** 8.4 ± 0.2*,## 8.4 ± 0.7**,## 9.0 ± 0.6**,## Left kidney weight/BW, mg/g 6.9 ± 0.1 6.9 ± 0.3 10.5 ± 0.3*** 8.4 ± 0.1# 7.7 ± 0.5### 9.3 ± 0.7*** Liver weight/BW, mg/g 42.5 ± 1.5 43.9 ± 1.2 59.7 ± 1.3** 52.1 ± 4.2* 59.6 ± 2.0*** 58.8 ± 3.7*** Lung weight/BW, mg/g 5.8 ± 0.2 5.9 ± 0.2 7.5 ± 0.4*** 6.6 ± 0.2 6.6 ± 0.2 7.3 ± 0.2*** Heart weight/BW, mg/g 4.9 ± 0.1 5.6 ± 0.6 5.9 ± 0.2 5.5 ± 0.3 4.8 ± 0.1 6.0 ± 0.4 Data are presented as mean ± SEM.
BW, body weight; D, diabetic; dh-3, -10, and -20, dh404 at 3, 10, and 20 mg/kg/day; ND, nondiabetic.
*P < 0.05, **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D+SO.
- Table 2
Metabolic parameters of nondiabetic and diabetic ApoE−/− mice treated with vehicle (SO) or dh404 at conclusion of 18-week study
ND+SO (n = 9) ND+dh-20 (n = 8) D+SO (n = 5) D+dh-3 (n = 7) D+dh-10 (n = 8) D+dh-20 (n = 9) HbA1c, % 4.3 ± 0.1 3.5 ± 0.1 13.2 ± 1.0 10.4 ± 1.9 10.6 ± 1.0 10.5 ± 1.4 HbA1c, mol/mol 23 ± 1.1 15 ± 1.1 121 ± 10.9*** 90 ± 20.8*** 92 ± 10.9*** 91 ± 15.3*** Blood glucose, mmol/L 8.9 ± 0.7 8.2 ± 0.4 22.8 ± 2.0*** 20.7 ± 2.9*** 23.9 ± 1.6*** 21.7 ± 2.6*** Total cholesterol, mmol/L 10.5 ± 0.6 14.0 ± 1.4 21.9 ± 1.3*** 14.4 ± 1.5## 15.2 ± 1.4## 17.6 ± 1.6**,# Triglycerides, mmol/L 1.9 ± 0.4 1.3 ± 0.3 7.3 ± 1.0*** 4.4 ± 0.8*,# 4.0 ± 0.4*,# 5.3 ± 0.9** HDL, mmol/L 2.2 ± 0.1 3.1 ± 0.3* 4.3 ± 0.3*** 2.7 ± 0.3## 3.1 ± 0.3*,# 3.2 ± 0.3*,# LDL, mmol/L 7.5 ± 0.4 10.4 ± 1.0 14.4 ± 0.9*** 9.8 ± 1.1# 10.3 ± 1.1 12.0 ± 1.1** Plasma AST, units/L 155 ± 34 103 ± 17 201 ± 82 153 ± 42 233 ± 74 187 ± 72 Plasma ALT, units/L 43 ± 8 29 ± 16 56 ± 17 50 ± 20 100 ± 39 51 ± 33 Water intake, mL/24 h 4.1 ± 0.4 0.7 ± 0.2 24.0 ± 1.6*** 17.1 ± 3.0*** 19.4 ± 2.2*** 16.6 ± 3.9*** Food intake, g/24 h 2.4 ± 0.2 3.2 ± 0.2 5.2 ± 0.4*** 3.8 ± 0.3* 4.4 ± 0.3*** 4.9 ± 0.5*** Urinary output, mL/24 h 1.0 ± 0.2 1.9 ± 0.2 22.5 ± 2.0*** 13.6 ± 2.7** 14.7 ± 2.3** 16.7 ± 4.1*** Data are presented as mean ± SEM.
D, diabetic; dh-3, -10, and -20, dh404 at 3, 10, and 20 mg/kg/day; ND, nondiabetic.
*P < 0.05, **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05, ##P < 0.01 vs. D+SO.
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