Combination therapy with artemether and enalapril improves type 1 diabetic nephropathy through enhancing antioxidant defense

Abstract

Previous studies have demonstrated that both artemether and enalapril are effective in treating diabetic nephropathy (DN). However, the effects and underlying mechanisms of their combination in treating DN remain unknown. The experimental DN model was induced by injecting streptozotocin (STZ) into male C57BL/6J mice. Mice were randomly allocated to the Type 1 diabetes control (T1D-ctrl), STZ, STZ + artemether (STZ + Art), STZ + enalapril (STZ + ACEi), or STZ + artemether + enalapril (STZ + Art + ACEi) group. The interventions lasted for 8 weeks. At the end of the experiment, related urine and serum biochemical values, such as urinary albumin excretion (UAE) and fasting blood glucose (FBG), were measured. In addition, blood pressure (BP) and kidney morphologic changes were also evaluated. The expression of oxidative stress related molecules, such as catalase, acetylated SOD2 (k68) and acetylated SOD2 (k122) in the kidney were measured. Results: combination therapy showed more pronounced effects in reducing UAE, FBG, and BP than any single drug. Typical diabetic kidney injuries, such as heavier kidney weight, and glomerular and tubular hypertrophy, were also further alleviated by combination therapy. Combination therapy also up-regulated the expression of catalase and down-regulated the expression of acetylated SOD2 (k68) and acetylated SOD2 (k122). Combination therapy with artemether and enalapril exhibited renoprotective effects in STZ-induced T1D mice superior to a single drug. The mechanism might be associated with their synergistic effects in enhancing antioxidant defense.

Keywords: ACEi; Diabetic nephropathy; artemether; combination; enalapril.

Figure 1

Effects of artemether, enalapril alone, or their combination on blood glucose, HbA1c, urine glucose and diabetic symptoms. A: Bar graphs representing the concentration of fasting blood glucose at 0, 2, 4, 6, 8 weeks post-treatment. B, C: Bar graphs representing the levels of HbA1c and urinary glucose at the end of the experiment. D-G: Bar graphs representing the quantification of food intake, water consumption, feces, and urine weight in all groups at the end of the experiment. n=7-8 per group. ***P<0.001 vs. the T1D-ctrl group; #P<0.05, ##P<0.01, and ###P<0.001 vs. the STZ group; ΔP<0.05, ΔΔP<0.01, and ΔΔΔP<0.001 vs. STZ + Art group; ♦♦P<0.01, and ♦♦♦P<0.001 vs. STZ + ACEi group.

Figure 2

Combination therapy with artemether and enalapril decreased blood pressure. A-C: Bar graphs denoting the quantification of systolic blood pressure, mean artery pressure, and diastolic blood pressure in different groups. n=8 per group. #P<0.05, ##P<0.01, and ###P<0.001 vs. the STZ group; ΔΔΔP<0.001 vs. STZ + Art group; ♦P<0.05, ♦♦P<0.01 and ♦♦♦P<0.001 vs. STZ + ACEi group.

Figure 3

Effects of artemether, enalapril alone, or their combination on biochemical measures. A, B: Bar graphs representing the levels of serum albumin and total protein. C-F: Bar graphs representing the levels of TG, LDL, HDL, and TC in different groups. n=8 per group. **P<0.01 and ***P<0.001 vs. the T1D-ctrl group; #P<0.05 and ##P<0.01 vs. the STZ group; ΔP<0.05 and ΔΔP<0.01 vs. STZ + Art group; ♦♦♦P<0.001 vs. STZ + ACEi group.

Figure 4

Effects of artemether, enalapril alone, or their combination on urine and serum measures related to kidney function. A-C: Urinary albumin excretion, urinary albumin to creatinine ratio, and urinary excretion of NAG at the end of the study in various groups. D, E: Blood creatinine and urea nitrogen concentration at the end of the study in each group. n=6-8 per group. ***P<0.001 vs. the T1D-ctrl group; #P<0.05, ##P<0.01, and ###P<0.001 vs. the STZ group; ΔΔP<0.01, and ΔΔΔP<0.001 vs. STZ + Art group; ♦P<0.05 and ♦♦♦P<0.001 vs. STZ + ACEi group.

Figure 5

Combination therapy with artemether and enalapril prevented diabetic kidney enlargement and attenuated glomerular and tubular injuries. A: Kidney weights in indicated groups at the end of the experiment. B-D: Bar graphs representing the GTA, GTV, and mesangial matrix area in each group. E-G: Bar graphs indicating the tubular cross-section area, tubular lumen area, and wall area in various groups. H: Representative PAS staining images for the glomeruli. Scale bars, 20 μm. I: Representative PAS stained images of the tubules. Scale bars, 20 μm. n=7-8 per group. *P<0.05, **P<0.01 and ***P<0.001 vs. the T1D-ctrl group; #P<0.05, ##P<0.01, and ###P<0.001 vs. the STZ group; ΔP<0.05 and ΔΔP<0.01 vs. STZ + Art group; ♦♦P<0.01 and ♦♦♦P<0.001 vs. STZ + ACEi group.

Figure 6

Combination therapy with artemether and enalapril regulated renal endogenous antioxidant protein expression. A: Western blot images of acetylated SOD2 (k122), acetylated SOD2 (k68), and catalase at 8 weeks post-treatment. B-D: Bar graphs indicating the fold change in the aforementioned protein expression after normalization to β-actin. E: Immunohistochemical staining images of acetylated SOD2 (k122), acetylated SOD2 (k68), and catalase in the kidney of different groups. Scale bars: 200 μm. n=4-8 per group. ***P<0.001 vs. the T1D-ctrl group; ##P<0.01 and ###P<0.001 vs. the STZ group; ♦♦P<0.01 vs. STZ + ACEi group.

Figure 7

Administration of artemether or enalapril alone or their combination showed no hepatotoxicity. A, B: Bar graphs representing the levels of serum ALT and AST. n=8 per group. ***P<0.001 vs. the T1D-ctrl group; ###P<0.001 vs. the STZ group.