Antiatherosclerotic and renoprotective effects of ebselen in the diabetic apolipoprotein E/GPx1-double knockout mouse

Abstract

Objective: To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.

Research design and methods: The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.

Results: Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.

Conclusions: Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

FIG. 1.

Ebselen reduces diabetes-associated atherosclerosis in the ApoE^−/−GPx1^−/− (dKO) aorta. Sudan IV-stained aortas from (A) nondiabetic, (B) diabetic, and (C) ebselen-gavaged diabetic dKO mice, 20 weeks after sham or streptozotocin-induced diabetes. Total and regional plaque is shown in D and E, respectively; Bars, mean ± SEM (n = 6–10 aortas/group). ***P < 0.001, **P < 0.01 vs. nondiabetic controls; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. diabetic dKO aortas. Abd, abdominal; D, diabetic; Eb, ebselen; ND, nondiabetic; Thor, thoracic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Ebselen attenuates ApoE^−/−GPx1^−/− (dKO) aortic mRNA expression after 10 weeks of STZ-induced diabetes. (A) Nox2, (B) VEGF, (C) CTGF, (D) MCP-1, and (E) VCAM-1 quantitative RT-PCR levels were expressed relative to nondiabetic dKO levels, which were arbitrarily assigned a value of 1. Bars, mean ± SEM (n = 7–9 aortas/group). ***P < 0.001, **P < 0.01, *P < 0.05 vs. nondiabetic dKO aortas; ###P < 0.001, ##P < 0.01 vs. diabetic dKO aortas. D, diabetic; Eb, ebselen; ND, nondiabetic.

FIG. 3.

Ebselen attenuates ApoE^−/−GPx1^−/− (dKO) aortic protein levels after 10 weeks of STZ-induced diabetes. Representative immunhistochemical staining for each protein is shown in supplementary online Fig. 2A–F. Quantitation of immunohistochemical staining within aortic sections is shown for (A) Nox2, (B) nitrotyrosine (NT), (C) 4-HNE, (D) VEGF, (E) CTGF, and (F) VCAM-1. Values are expressed relative to the nondiabetic dKO group which is arbitrarily assigned a value of 1. Bars, mean ± SEM n = 5–7 aortas/group; *P < 0.05 vs. nondiabetic dKO group; #P < 0.05 vs. diabetic dKO group. a.u., arbitrary units; D, diabetic; Eb, ebselen; NT, nitrotyrosine; ND, nondiabetic.

FIG. 4.

Ebselen attenuates structural and functional markers of nephropathy in the diabetic ApoE^−/−GPx1^−/− (dKO) kidney. A: Albuminuria is significantly reduced after 10 weeks of treatment in the diabetic dKO kidney, ##P < 0.01 vs. diabetic dKO, ***P < 0.001 and **P < 0.01 vs. nondiabetic counterparts, n = 4–8 urines/group, Bars, geometric mean ± error bars. B: Representative photomicrographs of PAS-stained glomeruli, (i) nondiabetic dKO, (ii) diabetic dKO, (iii) diabetic dKO+Eb. C: Ebselen significantly attenuated PAS staining after 10 and 20 weeks of treatment in diabetic dKO kidneys, ###P < 0.001 vs. diabetic dKO kidneys; ***P < 0.001 vs. nondiabetic age-matched dKO kidneys. D: PAS staining of kidneys showed significantly more damage in the diabetic dKO kidney versus diabetic ApoE^−/− controls (***P < 0.001 vs. diabetic ApoE^−/− kidneys), ***P < 0.001 vs. nondiabetic kidneys; n = 6–9 mice/group and at least 20 glomeruli/mouse. 10w, 10 weeks of diabetes; 20w, 20 weeks of diabetes; D, diabetic; Eb, ebselen; ND, nondiabetic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

The H₂O₂-mediated increase in nitrotyrosine (NT) is reduced by ebselen in the diabetic ApoE^−/−GPx^−/− (dKO) kidney. A–D: Representative photomicrographs of kidney tubules after 20 weeks of diabetes. (A) nondiabetic dKO, (B) diabetic dKO, (C) diabetic dKO + Eb, and (D) negative control which consisted of species-matched nonimmune IgG in place of primary antibody. E–H: Representative photomicrographs of kidney glomeruli after 20 weeks of diabetes. Nondiabetic dKO (E); Diabetic dKO (F); diabetic dKO + Eb (G); and negative control which consisted of species-matched nonimmune IgG in place of primary antibody (H). Quantitation of nitrotyrosine-stained tubules and glomeruli is shown in I and J, respectively. ***P < 0.001; diabetic dKO versus diabetic ApoE^−/− kidneys. ##P < 0.01 and ###P < 0.001 vs. diabetic dKO kidneys. Bars, mean ± SEM; n = 6–8 kidneys/group. a.u., arbitrary units; D, diabetic; Eb, ebselen; ND, nondiabetic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

The H₂O₂-mediated increase in Nox2 is reduced by ebselen in the diabetic ApoE^−/−GPx1^−/− (dKO) kidney. A: Quantitative RT-PCR analysis of Nox2 in ApoE^−/− and dKO kidneys after 20 weeks of diabetes; n = 8–10 kidneys/group. B: Representative photomicrographs of kidney glomeruli (i–iv) and tubules (v–viii); nondiabetic dKO (i and v); diabetic dKO (ii and vi); diabetic dKO+Eb (iii and vii); and negative control which consisted of species-matched nonimmune IgG in place of primary antibody (iv and viii). C and D: Quantitation of Nox2 protein within glomeruli and tubules, respectively. ***P < 0.001; **P < 0.01; *P < 0.05 diabetic dKO vs. diabetic ApoE^−/− kidneys; and versus nondiabetic counterparts. ##P < 0.01 and ###P < 0.001 vs. diabetic dKO kidneys. Bars, mean ± SEM; n = 20 glomeruli/kidney and 4–6 kidneys/group and n = 6 tubular fields/kidney and at least 5 kidneys/group. a.u., arbitrary units; D, diabetic; Eb, ebselen; ND, nondiabetic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

The H₂O₂-mediated increase in fibronectin is reduced by ebselen in the diabetic ApoE^−/−GPx1^−/− (dKO) kidney. A: Quantitative RT-PCR analysis of fibronectin in ApoE^−/− and dKO kidneys after 20 weeks of diabetes; n = 7–10 kidneys/group. B: Representative photomicrographs of kidney glomeruli (i–iv) and tubules (v–viii); nondiabetic dKO (i and v); diabetic dKO (ii and vi); diabetic dKO+EB (iii and vii); and negative control which consisted of species-matched nonimmune IgG in place of primary antibody (iv and viii). C and D: Quantitation of fibronectin protein within glomeruli and tubules, respectively. ***P < 0.001; **P < 0.01; *P < 0.05 and ###P < 0.001; ##P < 0.01; #P < 0.05 as indicated by horizontal bars. Bars, mean ± SEM; n = 20 glomeruli/kidney and 4–6 kidneys/group and n = 6 tubular fields/kidney and at least 5 kidneys/group. a.u., arbitrary units; D, diabetic; Eb, ebselen; ND, nondiabetic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 8.

Ebselen abrogates H₂O₂-mediated increases in (A and D) P-p38; (B and E) P-IKK, and (C and F) P-JNK protein in normal rat kidney cells. A representative gel with its internal α-tubulin control (Aii, Biv, and Cvi) is shown above the quantitation for each protein. Lane 1 = untreated cells; lane 2 = serum-starved (SS) cells for 4 h; lane 3 = DMSO-treated cells; lane 4 = DMSO + 1 mmol/l H₂O₂-treated cells; lane 5 = 1 mmol/l H₂O₂ treated cells; lane 6 = 0.03 μmol/l ebselen-treated cells; and lane 7 = 1 mmol/l H₂O₂+0.03 μmol/l ebselen-treated cells. Arrows point to the two isoforms of P-JNK. Phosphorylated protein was quantitated relative to total protein (total protein levels are shown in supplementary online Fig. 11) for each gene. *P < 0.05 and **P < 0.01 vs. control cells, DMSO, and serum-starved cells. #P < 0.05 Ebselen plus H₂O₂ versus H₂O₂-treated cells. Bars, mean ± SEM; n = 4 replicates/group.