Amiloride Improves Endothelial Function and Reduces Vascular Stiffness in Female Mice Fed a Western Diet

Figures

• 

  Figure 1. Effects of Western diet (WD) consumption and amiloride treatment on body composition. (A) Body fat percent in 20-week-old female mice fed either a control diet (CD) or a WD and treated (CDA, WDA) or not (CDC, WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Percent lean mass in the same mice cohorts. (C) Body weight at sacrifice (24-week-old) in the same mice cohorts. (D) Amount of retroperitoneal fat present at sacrifice in the same mice cohorts. (E) Amount of periovarian fat present at sacrifice in the same mice cohorts. Data are means ± SE of n = 4–13. *P < 0.05 vs. CDC, #vs. CDA.

• 

  Figure 2. Effects of Western diet (WD) consumption and amiloride treatment on kidney function, blood pressure, and glucose metabolism. (A) Daily Na+ excretion in 23-week-old female mice fed either a control diet (CD) or a WD and treated (CDA, WDA) or not (CDC, WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Proteinuria expressed as Protein (mg)/Creatinine (mg) in the same mice cohorts. (C) Mean arterial pressure (MAP) as determined via carotid artery catheterization in WDC and WDA 24-week-old female mice. (D) Homeostatic model assessment of insulin resistance (HOMA-IR) in the same mice cohorts as in (A). (E) Fasting circulating glucose in the same mice cohorts as in (A). Data are means ± SE of n = 3–13. *P < 0.05 vs. CDC, #vs. CDA, &vs. WDC.

• 

  Figure 3. Amiloride reduces vascular stiffness. (A) Pulse wave velocity (PWV) in 23-week-old female mice fed either a control (CD) or a Western diet (WD) and treated (CDA, WDA) or not (CDC, WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Endothelial stiffness as measured by atomic force microscopy in WDC and WDA female mice. (C) Endothelial stiffness as measured by atomic force microscopy in female mice fed WD and treated ex vivo with vehicle control (WD+V) or with amiloride (WD+A). Data are means ± SE of n = 5–13. *P < 0.05 vs. CDC, &vs. WDC or WD+V.

• 

  Figure 4. Amiloride improves endothelial function and reduces the distensibility of mesenteric arteries from female mice fed a Western diet (WD). (A) Percent acetylcholine-induced dilation of isolated cannulated and pressurized mesenteric arteries obtained from 24-week-old female mice fed a Western diet (WD) and treated (WDA) or not (WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Percent sodium nitroprusside-induced dilation of the same arteries as in (A). (C) Passive pressure diameter curves of the same mesenteric arteries as in (A). (D) Cross-sectional area (CSA) of the arterial wall from the same vessels as in (A). (E) Strain-stress relationships of mesenteric arteries from the same vessels as in (A). (F) Incremental elastic moduli of elasticity of the same mesenteric arteries as in (A). Data are means ± SE of n = 4–5. *P < 0.05 vs. WDC.

• 

  Figure 5. Amiloride reduces fibrosis and oxidative stress without altering EnNaC abundance in aortae of female mice fed a Western diet (WD). (A) Top panels are representative images (red = collagen), while the bottom panel represents the quantification of fibrosis as determined by picrosirius red staining in aortae from 24-week-old female mice fed a WD and treated (WDA) or not (WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Top panels are representative images (brown = 3NT), while the bottom panel represents the quantification of oxidative stress as measured by presence of 3-nitrotyrosine (3NT) in the intima (left bars), media (center bars), and adventitia (right bars) in aortae from the same mice cohorts as in (A). (C) Top panels are representative images (blue = elastin), while the bottom panel represents the quantification of medial thickness of aortae from the same mice cohorts as in (A). (D) Top panels are representative images (blue = nuclei, red = EnNaC), while the bottom panel represents the quantification of the presence of EnNaC in the endothelium of aortae from the same mice cohorts as in (A). Data are means ± SE of n = 4–7. *P < 0.05 vs. WDC.

• 

  Figure 6. Amiloride induces the arterialization of mesenteric arteries in female mice fed a Western diet (WD). (A) Top panels are representative images, while the bottom panel represents the quantification of medial F-actin content in mesenteric arteries from 24-week-old female mice fed a WD and treated (WDA) or not (WDC) with amiloride (1 mg/kg/day) in the drinking water. (B) Top panels are representative images, while the bottom panel represents the quantification of elastin content in the same mesenteric arteries as in (A). (C) Top panels are representative images, while the bottom panel represents the quantification of collagen content in the same mesenteric arteries as in (A). (D) Top panels are representative images, while the bottom panel represents the quantification of EnNaC presence in the same mesenteric arteries as in (A). Data are means ± SE of n = 3–4. *P < 0.05 vs. WDC. All images were contrast-enhanced equally to improve their visualization in print. All analyses were performed using raw data.

• 

  Figure 7. Effects of amiloride treatment on the characteristics of mesenteric artery internal elastic lamina fenestrae in Western diet (WD) fed female mice. (A) Representative image of the internal elastic lamina and its fenestrae of a mesenteric artery obtained from a 24-week-old female mouse fed a WD and not treated with amiloride. (B) Representative image of the internal elastic lamina and its fenestrae of a mesenteric artery obtained from a 24-week-old female mouse fed a WD and treated with amiloride. (C) Number of fenestrae per area (400 × 1,024 pixels) in the internal elastic lamina of 24-week-old female mice fed a WD and treated or not (WDA, WDC, respectively) with amiloride (1 mg/kg/day) in the drinking water. (D) Mean area per fenestra in the same vessels as in (C). Data are means ± SE of n = 3–4. All images were contrast-enhanced equally to improve their visualization in print. All analyses were performed using raw data.

• 

  Figure 8. Ex vivo exposure to amiloride does not increase the vasodilatory responses to flow of mesenteric arteries obtained from endothelium-specific mineralocorticoid receptor knock out female mice (ECMR−/−). Isolated cannulated and pressurized mesenteric arteries from ECMR−/− female mice fed a Western diet (WD) were incubated with amiloride (1 uM) for 20 min, pre-constricted with 10−5 M phenylephrine (Phe) and then exposed to increasing levels of intraluminal flow while maintaining mean intravascular pressure at 70 mmHg in order to determine flow-mediated vasodilatory effects. The vasodilatory effects of flow on ECMR−/− arteries treated with vehicle control (NoA) that were published previously (red line)3 are included for comparison. Data are means ± SE of n = 4–5.