Lipid Profiles of Urinary Extracellular Vesicles Released during the Inactive and Active Phases of Aged Male Mice with Spontaneous Hypertension

Figures

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  Figure 1. Tail cuff systolic blood pressure measurements of 18-month-old male mice during the inactive (AM) phase (IP) and active (PM) phase (AP). (A) Summary plot of systolic blood pressure in aged male mice taken during the inactive phase (8 AM) or during the active phase (8 PM). (B) Summary plot of systolic blood pressure in these mice at the same time points but after the mice were injected with benzyl amiloride. (C) Summary plot of systolic blood pressure during the inactive phase (8 AM) before and after the mice were injected with benzyl amiloride. (D) Summary plot of systolic blood pressure during the active phase (8 PM) before and after the mice were injected with benzyl amiloride. A Wilcoxon rank sum test was performed to compare the two groups. n = 7 mice in each group. The open circles represent individual data points from the AP group and the open triangles represent individual data points from the IP group. * Represents a p-value of ≤0.05.

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  Figure 2. Urinary electrolyte measurements of 18-month old male mice during the inactive and active phases. (A) Summary plot of sodium excreted in the urine during the active and inactive phases from aged male mice (B) Summary plot of potassium excreted in the urine during the active and inactive phases from aged male mice. A Wilcoxon rank sum test was performed to compare the two groups. n = 7 in each group. The open circles represent individual data points from the AP group and the open triangles represent individual data points from the IP group. * Represents a p-value of ≤0.05.

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  Figure 3. Concentration and Western blot characterization of EVs isolated from urine samples collected during the inactive and active phase of spontaneously hypertensive male C57B6 mice. (A) Summary plot of uEV concentration from urine produced during the active phase and collected at 8 AM and from urine produced during the inactive phase and collected at 8 PM. (B) Western blot (WB) analysis of multiple EV markers including GAPDH, syntenin, flotillin, and caveolin-1. A Wilcoxon rank sum test was performed to compare the two groups. n = 7 EV preps in each group. The open circles represent individual data points from the AP group and the open triangles represent individual data points from the IP group.

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  Figure 4. Phosphatidylethanolamine levels in uEVs isolated from urine collected during the inactive and active phases of spontaneously hypertensive aged male mice. (A) Volcano plot showing all PE’s increased or decreased in the EVs from the active phase (AP) group compared to the inactive phase (IP) group and all other PE’s that did not show a statistically significant difference between the two groups. The fold change threshold was set to 2.0 and the p-value threshold was set to 0.05. Data were normalized to the median with Pareto scaling and the plots were made in MetaboAnalyst software. (B) Normalized plots showing 7 PE’s that are significantly increased in EVs isolated from urine samples collected during the AP compared to the IP. (C) Normalized plots showing 28 PE’s that are significantly decreased in EVs from the AP group compared to the IP group. n = 7 samples per group. EVs isolated from AP urine are in red and EVs isolated from IP urine are in green. The concentration of each lipid on the y-axis is given in μM.

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  Figure 5. Triacylglycerol levels in uEVs isolated from urine collected during the inactive and active phases of spontaneously hypertensive aged male mice. (A). Normalized plots showing 3 TAGs that are significantly increased in EVs isolated from urine samples collected during the active phase (AP) compared to the inactive phase (IP). (B). Normalized plots showing 6 TAGs that are significantly decreased in EVs from the AP group compared to the IP group. Data were normalized to the median with Pareto scaling and the plots were made in MetaboAnalyst software. n = 7 samples per group. EVs isolated from AP urine are in red and EVs isolated from IP urine are in green. The concentration of each lipid on the y-axis is given in μM.

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  Figure 6. Phosphatidylcholine levels in uEVs isolated from urine collected during the inactive and active phases of spontaneously hypertensive aged male mice. (A) Normalized plots showing 3 PC’s that are significantly increased in EVs isolated from urine samples collected in the active phase (AP) compared to the inactive phase (IP). (B) Normalized plots showing 3 PC’s that are significantly decreased in EVs from the AP group compared to the IP group. Data were normalized to the median with Pareto scaling and the plots were made in MetaboAnalyst software. n = 7 samples per group. EVs isolated from AP urine are in red and EVs isolated from IP urine are in green. The concentration of each lipid on the y-axis is given in μM.

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  Figure 7. Amiloride-sensitive transepithelial current measurements in mpkCCD cells treated with 2 × 107 particle/mL of EVs isolated from the urine collected during the inactive phase or active phase of spontaneously hypertensive mice. * Represents a p-value of ≤0.05, ** represents a p-value of <0.01.

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  Figure 8. Sucrose density gradients showing the distribution of ENaC alpha subunit to lipid rafts after treating mpkCCD cells with uEVs from the active phase compared to the inactive phase. mpkCCD cells were challenged with uEVs from the inactive phase (A) or the active phase (B) before isolating lipid rafts and non-lipid rafts. Representative Western blots for ENaC alpha subunit protein expression in each fraction. Caveolin 1 (Cav1) is present in the light density gradient fractions corresponding to lipid rafts, while the mu-2 protein is present in non-lipid raft associated fractions. n = 2 independent experiments.