Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice

Abstract

Background/aims: Canonical Wnt signaling is involved in oxidative stress, vasculopathy and diabetes mellitus but its role in diabetic renal microvascular dysfunction is unclear. We tested the hypothesis that enhanced canonical Wnt signaling in renal afferent arterioles from diabetic mice increases reactive oxygen species (ROS) and contractions to endothelin-1 (ET-1).

Methods: Streptozotocin-induced diabetes or control C57Bl/6 mice received vehicle or sulindac (40 mg·kg-1·day-1) to block Wnt signaling for 4 weeks. ET-1 contractions were measured by changes of afferent arteriolar diameter. Arteriolar H2O2, O2 -, protein expression and enzymatic activity were assessed using sensitive fluorescence probes, immunoblotting and colorimetric assay separately.

Results: Compared to control, diabetic mouse afferent arteriole had increased O2- (+ 84%) and H2O2 (+ 91%) and enhanced responses to ET-1 at 10-8 mol·l-1 (-72±4% of versus -43±4%, P< 0.05) accompanied by reduced protein expressions and activities for catalase and superoxide dismutase 2 (SOD2). Arteriolar O2 - was increased further by ET-1 and contractions to ET-1 reduced by PEG-SOD in both groups whereas H2O2 unchanged by ET-1 and contractions were reduced by PEG-catalase selectively in diabetic mice. The Wnt signaling protein β-catenin was upregulated (3.3-fold decrease in p-β-catenin/β-catenin) while the glycogen synthase kinase-3β (GSK-3β) was downregulated (2.6-fold increase in p-GSK-3β/ GSK-3β) in preglomerular vessels of diabetic mice. Sulindac normalized the Wnt signaling proteins, arteriolar O2 -, H2O2 and ET-1 contractions while doubling microvascular catalase and SOD2 expression in diabetic mice.

Conclusion: Increased ROS, notably H2O2 contributes to enhanced afferent arteriolar responses to ET-1 in diabetes, which is closely associated with Wnt signaling. Antioxidant pharmacological strategies targeting Wnt signaling may improve vascular function in diabetic nephropathy.

Keywords: Afferent arteriole; Canonical Wnt signaling; Diabetes mellitus; Endothelin-1; Reactive oxygen species.

Fig. 1.

Meant ± SEM values (n =5–6) for ET-1 responses of afferent arterioles from control mice in the absence (black diamonds with black line) or presence of PEG-SOD (200 units mL⁻¹) (A) or PEG-catalase (1000 units mL⁻¹) (B) or Sulindac (40 mg·kg⁻¹ day⁻¹ for 4 weeks) (C) (blue triangles with blue line), diabetic mice in the absence (grey squares with grey line) or presence of PEG-SOD (A) or PEG catalase (B) or Sulindac) (C) (red crosses with red line). Data are shown for changes in luminal dimeters of afferent arterioles in response to ET-1 (10⁻¹² - 10⁻⁸ mol·l⁻¹). ANOVA, analysis of variance; ET-1, endotheline-1; PEG, polyethylene glycol; SOD, superoxide dismutase; DM, diabetes mellitus.

Fig. 2.

Mean ± SEM values (n=5) for O₂^.− and H₂O₂ in the perfused individual afferent arteriole (A and B) or in the isolated preglomerular arterioles (C and D) from control mice (open boxes), diabetic mice (blue filled boxes) and sulindac treated diabetic mice (grey filled boxes). Data are shown as the basal levels of ethidium:dihydroethidium (E:DHE) fluorescence ratio for O₂^.−, H₂DCFDA fluorescence for H₂O₂, their changes in response to ET-1 (10⁻⁸ mol·l⁻¹) in the perfused individual arteriole and units (or μmol)·mg-l protein for their concentrations in the isolated preglomerular arterioles. Comparing groups: * P<0.05; ‡ P< 0.001.

Fig. 3.

Time course of changes in luminal diameter of perfused afferent arterioles (n=5) from normal control (without injection, black diamonds with solid line), vehicle control (7 days after injection of citrate buffer, grey diamonds with grey line) and diabetic mice (7 days after injection of STZ, blue triangles with blue line) by bath addition of H₂O₂ (10 μmol·l⁻¹). ANOVA, analysis of variance.

Fig. 4.

Mean ± SEM values (n=5) for the ratio of phosphorylated protein p-GSk-3β/GSk-3β (A) and p-β-catenin/β-catenin (B) in preglomerular arterioles from control mice (open boxes), diabetic mice (blue filled boxes) and Sulindac treated diabetic mice (grey filled boxes). Comparing groups: * P< 0.05; ‡ P< 0.001.

Fig. 5.

Mean ± SEM values (n=5) for protein expression of catalase, SOD1 and SOD2 in preglomerular arterioles from control mice (open boxes), diabetic mice (blue filled boxes) and Sulindac treated diabetic mice (grey filled boxes). Comparing groups: ‡ P< 0.001.

Fig. 6.

Mean ± SEM values (n=5) for enzymatic activity of total SOD (A) and catalase (B) in preglomerular arterioles from control mice (open boxes), diabetic mice (blue filled boxes) and Sulindac treated diabetic mice (grey filled boxes). Comparing groups: * P< 0.05; ‡ P< 0.001.