Studies of salt and stress sensitivity on arterial pressure in renin-b deficient mice

Figures

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  Fig 1. Experimental protocol. The assessment of salt sensitivity (Experiment 1) and susceptibility to restraint-stress (Experiment 4) were performed in the same animals. Male Ren-bNull or wildtype (WT) littermate controls were instrumented with a radiotelemetry catheter and housed in a quiet environment for blood pressure and heart rate measurements. In experiment 1, baseline arterial blood pressure and heart rate were recorded for 3 days under a standard salt diet (SD, 0.8% NaCl). Subsequently, the diet was switched to a chow containing low sodium (LS, 0.02% NaCl), and data were acquired for 7 additional days. Finally, the diet was switched from low to high salt containing diet (HSD, 4% NaCl), and data were acquired for 7 additional days. To study responses to acute restraint stress in both HSD and LSD, the animals were continued on HSD for an additional week. By the end of the second week on HSD, mice were placed to an acrylic restrainer to assess the acute cardiovascular responses to restraint-stress. Next, mice were fed LSD for two weeks and the acute responses to restraint-stress were reassessed again.

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  Fig 2. Cohort analysis of arterial blood pressure. Reanalysis and comparison of the telemetric blood pressure in 5 independent cohorts of Ren-bNull and wildtype (WT) mice. Summary of the systolic blood pressure (SBP) during the light cycle is shown. This data was previously reported in references [22] and [30]. *P<0.05 vs WT by unpaired t-test.

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  Fig 3. Blood pressure response to salt. Effect of salt intake on systolic blood pressure (SBP), mean blood pressure (MBP), diastolic blood pressure (DBP), and heart rate (HR) in male Ren-bNull mice and wildtype littermate controls (WT) housed in a quiet environment. Following a 3-day baseline radiotelemetric data collection period, the diet was switched from a standard diet to a chow containing 0.02% NaCl (TD08290, LSD). Data were acquired for 7 days. Subsequently, mice were challenged with a diet containing 4% NaCl (TD03095, HSD), and data were collected for 7 additional days. Twenty-four-hour averaged A) SBP, B) DBP, C) MBP, and D) HR were expressed as mean ± standard error of the mean (top panel). For purposes of clarity and transparency, the individual points for weekly averaged E) SBP, F) DBP, G) MBP, and H) HR data were also plotted (bottom panel). Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons procedure. Adjusted p<0.05 was considered significant. *p<0.05 vs WT at baseline, #p<0.05 vs Ren-bNull at baseline, Φp<0.05 vs WT fed LSD, Ψp<0.05 vs Ren-bNull fed LSD.

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  Fig 4. Renal function. Urine concentrating capacity was assessed in male and female wildtype (Ren-b+/+), heterozygous Ren-b knockout (Ren-b+/-) and homozygous Ren-b knockout mice (Ren-b-/-). Urine was first collected for 60 minutes post-hypotonic saline challenge (500 microliters of 0.03 mol/L NaCl; intraperitoneal). Subsequently, the same procedure was performed for a hypertonic saline challenge (500 microliters of 0.3 mol/L NaCl; intraperitoneal). *p<0.05 vs same genetic group on hypotonic challenge.

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  Fig 5. Blood pressure response to restraint stress (HSD). Acute responses to restraint-induced blood pressure (BP) and tachycardia in male Ren-bNull mice and wildtype littermate controls (WT) fed high salt diet. First, telemetric recordings were obtained from high salt diet-fed mice in their home cages at baseline. Following 10 minutes of recording, mice were immediately inserted into an acrylic restrainer and telemetric data were acquired for 10 minutes. One-minute recording was averaged, and the systolic BP, diastolic BP, mean BP, and heart rate (A, B, C, and D, respectively) was expressed as mean±standard error of the mean. For purposes of clarity and transparency the individual 10 minutes systolic BP and heart rate (E and F, respectively) average of each animal under baseline and restrained conditions was plotted. Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons procedure. Adjusted p<0.05 was considered significant.

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  Fig 6. Blood pressure response to restraint stress (LSD). Acute responses to restraint-induced blood pressure (BP) and tachycardia in male Ren-bNull mice and wildtype littermate controls (WT) fed low salt diet. First, telemetric recordings were obtained from low salt diet-fed mice in their home cages at baseline. Following 10 minutes of recording, mice were immediately inserted into an acrylic restrainer and telemetric data were acquired for 10 minutes. One-minute recording was averaged, and the systolic BP, diastolic BP, mean BP, and heart rate (A, B, C, and D, respectively) was expressed as mean±standard error of the mean. For purposes of clarity and transparency the individual 10 minutes systolic BP and heart rate (E and F, respectively) average of each animal under baseline and restrained conditions was plotted. Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons procedure. Adjusted p<0.05 was considered significant.

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  Fig 7. A) Blood glucose was determined every 15 minutes before, during, and after restraint-stress in Ren-bNull and wildtype littermates (WT) males and females. A group of unrestrained Ren-bNull and WT controls, which were kept in their home cages was included. B) The area under the curve (AUC) was calculated and the individual values were plotted. By the end of the protocol the animals were decapitated, and trunk blood was collected for renin (C) and corticosterone (D) determinations. *P<0.05 vs unrestrained WT and #P<0.05 vs unrestrained Ren-bNull.

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  Fig 8. Using quantitative reverse transcription PCR total renin expression was measured in A) the rostral ventrolateral medulla (RVLM) and B) the whole hypothalamus. C and D) In the adrenal glands, total renin and renin-a were measured. The wildtype (WT) and Ren-bNull mice were fed a normal salt diet. The data were analyzed by unpaired t-test. *P<0.05 vs WT.