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

Pablo Nakagawa; Javier Gomez; Ko-Ting Lu; Justin L Grobe; Curt D Sigmund

doi:10.1371/journal.pone.0250807

. 2021 Jul 28;16(7):e0250807. doi: 10.1371/journal.pone.0250807

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

Pablo Nakagawa ^1,^*, Javier Gomez ¹, Ko-Ting Lu ¹, Justin L Grobe ¹, Curt D Sigmund ¹

Editor: Michael Bader²

PMCID: PMC8318244 PMID: 34319999

Abstract

Excessive sodium intake is known to increase the risk for hypertension, heart disease, and stroke. Individuals who are more susceptible to the effects of high salt are at higher risk for cardiovascular diseases even independent of their blood pressure status. Local activation of the renin-angiotensin system (RAS) in the brain, among other mechanisms, has been hypothesized to play a key role in contributing to salt balance. We have previously shown that deletion of the alternative renin isoform termed renin-b disinhibits the classical renin-a encoding preprorenin in the brain resulting in elevated brain RAS activity. Thus, we hypothesized that renin-b deficiency results in higher susceptibility to salt-induced elevation in blood pressure. Telemetry implanted Ren-b^Null and wildtype littermate mice were first offered a low salt diet for a week and subsequently a high salt diet for another week. A high salt diet induced a mild blood pressure elevation in both Ren-b^Null and wildtype mice, but mice lacking renin-b did not exhibit an exaggerated pressor response. When renin-b deficient mice were exposed to a high salt diet for a longer duration (4 weeks), there was a trend for increased myocardial enlargement in Ren-b^Null mice when compared with control mice, but this did not reach statistical significance. Multiple studies have also demonstrated the association of environmental stress with hypertension. Activation of the RAS in the rostral ventrolateral medulla and the hypothalamus is required for stress-induced hypertension. Thus, we next questioned whether the lack of renin-b would result in exacerbated response to an acute restraint-stress. Wildtype and Ren-b^Null mice equally exhibited elevated blood pressure in response to restraint-stress, which was similar in mice fed either a low or high salt diet. These studies suggest that mechanisms unrelated to salt and acute stress alter the cardiovascular phenotype in mice lacking renin-b.

Introduction

High blood pressure is a leading cause of complications from heart disease, stroke, and kidney disease. Importantly, it has been shown that in both normotensive and hypertensive subjects, increased sensitivity to sodium increases the risk for cardiovascular mortality and morbidity [1]. The increased prevalence of hypertension in the last century has been attributed in part to the higher consumption of dietary salt [2]. Moreover, there is strong evidence in animal models and human for a causative role of dietary sodium intake in the development of hypertension [2–4]. Dietary sodium reduction in combination with the DASH (dietary approaches to stop hypertension) diet is a powerful approach to control blood pressure in hypertensive patients [5,6].

According to Guyton and Coleman’s hypothesis, salt-sensitive hypertension can develop when there is an impaired excretory ability of the kidney that shifts the relation between sodium excretion and arterial pressure toward higher values [7]. Recently, Fujita et al. identified a key mechanism implicating renal β2-sympathetic stimulation contributing to the development of salt-sensitive hypertension [8]. Numerous studies have identified neurogenic actions of elevated salt intake [9,10]. For instance, elevated dietary sodium intake sensitizes sympathetic neurons in response to injection of L-glutamate into the rostral ventrolateral medulla [11]. In addition to the effects on arterial pressure, high salt diet can also adversely affect several organs including the vasculature, heart, and kidneys. Interestingly, some of these effects appear to be independent of blood pressure status [12].

The renin-angiotensin system (RAS) is recognized as a powerful circulating hormone system regulating blood pressure and sodium homeostasis. Although high salt intake suppresses the circulating classical RAS, the deterioration of renal function in sodium loaded animals can be attenuated by the blockade of the RAS [13]. Thus, it has been proposed that activation of a locally activated RAS within tissues, which acts independently of the circulating RAS, can be of major importance in the regulation of the angiotensin (Ang) II generation in the kidney, brain, vasculature, and heart [14–18]. Renin is the rate-limiting step of Ang II synthesis and exists in at least two isoforms. The classical renin isoform encoding preprorenin termed “renin-a” is primarily expressed by the juxtaglomerular cells of the kidney and is released into the circulatory system in response to a number of stimuli. On the contrary, the novel alternative renin isoform, termed “renin-b”, is mainly expressed in the brain, heart, and adrenal gland [19–21]. Current evidence suggests that renin-b is unlikely to be secreted as it lacks a secretory peptide. Our previous data suggest that renin-b maintains brain RAS activity at normal physiological levels by suppressing the classical renin isoform and that the genetic removal of renin-b triggers a mild increase in blood pressure and sympathetic nerve activity [22–24]. The sustained suppression of the brain RAS by renin-b in healthy individuals may have important implications in maintaining the cardiovascular homeostasis during different hypertension-triggering stimuli such as high salt diet and other environmental stressors. Blood pressure elevation induced by the exposure to physical stress can be ameliorated when the brain RAS is suppressed [25,26]. Given the rationale that hypertension-prone animal models exhibit a greater increase in blood pressure and heart rate in response to restraint-stress and air-jet stress [27,28], we hypothesized that mice lacking renin-b would be more responsive to an acute environmental stressor as consequence of brain RAS disinhibition.

Methods

Animals

All experiments were conducted using mice lacking exon 1b of the renin gene on the C57BL/6 genetic background as previously described [22]. Experimental animals were generated by intercrossing heterozygotes to generate homozygous control wildtypes (WT) and homozygous Ren-b^Null mice. Only males were used except where indicated. Males were preferred in this study because we previously observed that Ren-b^Null males exhibit an exacerbated phenotype compared to females [23]. A room with low levels of noise and traffic was designated for these experiments. Animals received standard laboratory chow (NIH-31 Modified Open Formula Mouse/Rat sterilizable diet; Teklad catalog # 7913; Harlan Teklad) until exposure to low and high salt diets. All experiments were conducted with the approval of the University of Iowa and Medical College of Wisconsin Animal Care and Use Committees and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Blood pressure measurements

Twenty-four-hour systolic, diastolic, and mean blood pressures and heart rate telemetric data were acquired by radiotelemetry. Telemetry implantation surgeries and telemetric data acquisition were performed by a surgeon who was blinded to genotype. Anesthesia was induced with ketamine (90 mg/kg i.p.)/xylazine (5 mg/kg i.p.) to implant the telemetry catheters. Then, a 1–2 cm incision was made to expose the internal carotid artery. A TA11PA-C10 radiotelemetry catheter (DSI, New Brighton, MN) was inserted in the left carotid artery and advanced into the transverse aorta. The catheter was secured with three sutures and the incision was sutured with 6–0 surgical silk. A triple antibiotic ointment, Vetropolycin (Dechra Pharmaceuticals PLC, Northwich, United Kingdom), was applied to the wound. Flunixin (2.5 mg/kg, i.p.) was immediately applied after the surgery and again 24 hours later. Then, the animals were housed in a cage placed on a heating pad overnight with easy access to food and water. Once awake from the insertion of the radiotelemetry implant procedure, the animals were housed individually in a regular cage placed on top of the telemetry receiver plate with free access to water and a standard chow diet containing 0.3% Sodium. Baseline blood pressure and heart rate data were acquired for 3 days after 10 days of post-surgical recovery. Blood pressure and heart rate data were recorded for 10 seconds every 5 minutes for 24 hours and averaged daily, hourly, and during the light (5 AM to 5 PM) and dark phase (5 PM to 5 AM).

Assessment of salt sensitivity

Experiment 1

Telemetry implanted mice underwent 3-day baseline blood pressure and heart rate data acquisition under a normal salt diet containing 0.8% NaCl (NIH-31 Modified Open Formula Mouse/Rat sterilizable diet; Harlan Teklad). The standard diet containing normal salt was then replaced with a salt-deficient chow diet containing 0.01–0.02% sodium (TD08290, Envigo, Indianapolis, IN). Data were acquired for 7 days on a low salt diet. Then, the diet was switched to a high salt chow containing 4% NaCl (TD03095, Envigo, Indianapolis, IN) and data were acquired for 7 additional days (Fig 1).

Fig 1 — The assessment of salt sensitivity (Experiment 1) and susceptibility to restraint-stress (Experiment 4) were performed in the same animals. Male Ren-b^Null 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.

Experiment 2

Renal concentrating response to acute hypo and hyperosmotic load was assessed in a cohort of male and female WT, heterozygous, and Ren-b^Null mice. Mice were forced to empty the bladder by immobilizing them. Then, hypotonic saline (500 microliters of 0.03 mol/L NaCl; intraperitoneal) was applied and the animal was placed in a 1 L beaker for 60 minutes. Subsequently, the same procedure was performed for a hypertonic saline challenge (500 microliters of 0.3 mol/L NaCl; intraperitoneal). Urine was collected from the beaker and the osmolality was assessed using a freezing point depression multi-sample osmometer (OsmoPro, Advanced Instruments, Norwood, MA). These experiments were performed from 9 AM to 12 PM.

Experiment 3

A separate cohort of mice without telemeters were placed in metabolic cages for urine collection at baseline, under a standard chow diet (0.8% NaCl), and after 4 weeks on a high salt diet (4% NaCl). Food and water intake, urinary volume, and protein excretion were assessed. Protein concentration was measured using a commercially available protein determination kit from Thermo Scientific (catalog # 23225), which is based on the bicinchoninic acid method. By the end of week 4, mice were anesthetized with pentobarbital and blood samples were obtained from vena cava using a heparinized syringe for measurement of plasma renin. Subsequently, mice were sacrificed, and kidneys, heart, liver, spleen, and lungs were collected and weighed.

Responses to restraint stress in mice fed high or low salt diets

Experiment 4

Telemetry implanted mice tested for salt-sensitivity in experiment 1 were subsequently subjected to acute experiments to assess sensitization to restraint-stress (Fig 1). First, we evaluated the cardiovascular responses to an acute immobilization protocol. WT and Ren-b^Null mice fed high salt diet were placed for 10 minutes in a restrainer, while telemetric blood pressure and heart rates were monitored. Then, the diet was switched back to a low salt diet and the same procedure was performed 14 days later. Baseline telemetric blood pressure and heart rate were acquired for 10 minutes before mice were placed in the restrainer. Once in the restrainer, data were acquired for 10 additional minutes. A separate cohort of males and females fed with a normal salt diet was used to study the metabolic responses to restraint-stress. Blood glucose was measured at baseline before restraint-stress and induced a restrain-stress for 30 minutes. Finally, mice were released to the home cage and blood glucose was measured again during recovery. Every 15 minutes blood samples were obtained from tail veins in conscious mice and blood glucose levels were determined using a glucometer (One Touch Ultra 1, LifeScan, Inc.). Mice were sacrificed by decapitation at 30 minutes post-recovery phase and blood was collected for renin and glucocorticoid determinations.

Plasma renin measurements

Heparinized blood was collected in 1.5 ml tubes from the inferior vena cava in anesthetized mice or trunk after decapitation. Blood samples were centrifuged at 4000 rpm for 2 min at 4° C to isolate plasma. Plasma renin was measured using a commercially available Sandwich-based ELISA kit (Raybiotech, Peachtree Corners, GA) following the manufacturer’s recommended instructions. Samples were processed and analyzed by a blinded operator.

Expression of renin in the central nervous system and adrenal glands

Experiment 5

Wildtype and Ren-b^Null mice fed a standard chow diet (0.8% NaCl) were sacrificed by injecting pentobarbital overdose (150 mg/kg). The animals were decapitated, the brains were excised from the skull and the adrenal glands collected and frozen in liquid nitrogen. The hypothalamus was carefully dissected and immediately immersed in liquid nitrogen. Some additional brains were embedded in Tissue-Tek OCT compound (Fisher Scientific, Waltham, MA) for subsequent rostral ventrolateral medulla (RVLM) sample collection using the puncture technique. With the help of a polypropylene pellet pestle the whole hypothalamic tissue, the RVLM punches, or adrenal glands were homogenized in TRIzol reagent (Thermo Fisher Scientific, Waltham, NA). The total mRNA was isolated with a Purelink RNA Mini kit (Invitrogen, Carlsbad, CA) and eluted in 30 microliters of RNase free water. Next, five hundred nanograms of total RNA was reverse transcribed using an iScript kit (Biorad, Hercules, CA). In the hypothalamic and RVLM samples, a real-time PCR was performed utilizing TaqMan probes and Fast Advanced Master mix (catalog # 4444557) from Applied Biosciences following the manufacturer’s protocol. The catalog numbers of Taqman probes were Ren1: Mm02342887_mH and Gapdh: Mm99999915_g1. In adrenal gland samples, a real-time PCR was performed using Applied Biosystems SYBR Green master mix and the following primer sets:

Ren1

Forward: 5’-TCCCGGACAGAAGGCATTTTC-3’
Reverse: 5’-TGTGTCACAGTGATTCCACCCACA-3’

Renin-a

Forward: 5’-GCACCTTCAGTCTCCCAACAC-3’
Reverse: 5’-TCCCGGACAGAAGGCATTTTC-3’

18s

Forward: 5’-CGCTTCCTTACCTGGTTGAT-3’
Reverse: 5’-GAGCGACCAAAGGAACCATA-3’

The data analysis was performed using the Livak method [29].

Statistical analysis

All data are presented as mean±SEM. Data were analyzed using GraphPad Prism software. All telemetric data, parameters derived from metabolic cage studies and osmotic challenge tests, plasma glucose, renin and corticosterone measurements were analyzed using two-way ANOVA with repeated measures, followed by Sidak’s multiple comparison procedures. Tissue weights were analyzed using unpaired t-test. A P value of less than 0.05 was considered significant. Individual data points were plotted in dot/whisker plots if possible.

Results

Retrospective analysis of the arterial blood pressure in Ren-b^Null cohorts

Retrospective analysis of blood pressure in five cohorts of Ren-b^Null and WT mice (listed in order of experimentation) indicated that mice lacking Ren-b exhibit a high degree of variability in blood pressure (range = 27.7 mmHg, Fig 2). The range of blood pressure in control mice was much smaller (5.5 mmHg). We hypothesized that these differences might be attributed to environmental factors as a review of conditions under which the cohorts were studied revealed that cohorts 1, 2, 3, and 4 were housed in a large room with an elevated level of noise and traffic (data collected between March 2014 and October 2015 [22]), while mice in cohort 5 were housed in a smaller and newer quiet animal facility located in a different building (data collected from 2/2017 to 11/2017 [30]). Cohorts 2 and 4 were exposed to an additional stress as they were subjected to a surgical implantation of a stainless-steel intracerebroventricular cannula for subsequent acute administration of drugs. These data were previously reported [22,30]. Cohorts 3 and 4 were subjected to extensive animal handling during experiments. Noise, post-surgical stress and animal handling appeared associated with an exacerbated blood pressure increase in Ren-b^Null mice. Therefore, the aim of this study was to elucidate whether the manifestation of cardiovascular and renal phenotypes in Ren-b^Null mice are due to increased sensitivity of these animals to external stressors such as environmental stimuli (i.e., noise, diet, and other psychological stresses such as restraint or handling).

Fig 2 — Reanalysis and comparison of the telemetric blood pressure in 5 independent cohorts of Ren-b^Null 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.

Effect of high salt diet on blood pressure in Ren-b^Null mice

Wildtype and Ren-b^Null mice fed a normal salt diet were instrumented with telemetric implants and allowed to recover for 10 days in a quiet environment. As shown in Fig 3, baseline blood pressure was first measured for 3 days under a normal salt diet containing 0.3% sodium. Then, the diet was replaced from normal to a low salt diet containing chow (Na 0.01–0.02%) and blood pressure recordings continued for 7 additional days. Finally, mice were fed high-salt diet chow (NaCl 4%) for the last 7 days. The low salt diet had no effects on systolic blood pressure, diastolic blood pressure, mean blood pressure or heart rate compared with the baseline diet (Fig 3). Blood pressure was increased in both wildtype and Ren-b^Null mice in response to the high salt diet when compared with the baseline diet (P<0.05) or low salt diet (P<0.05). Heart rate was not altered. Contrary to our hypothesis, there was no difference in blood pressure or heart rate comparing wildtype and Ren-b^Null mice in any diet.

Fig 3 — Effect of salt intake on systolic blood pressure (SBP), mean blood pressure (MBP), diastolic blood pressure (DBP), and heart rate (HR) in male Ren-b^Null 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-b^Null at baseline, ^Φp<0.05 vs WT fed LSD, ^Ψp<0.05 vs Ren-b^Null fed LSD.

To evaluate the capacity of Ren-b^Null mice to excrete a sodium load, mice were injected with either intraperitoneal hypo- or hyperosmotic NaCl solutions (0.5 ml of 0.03 and 0.3 mol/L NaCl, respectively). As expected, hyper-osmotic saline increased urinary osmolality compared with hypoosmotic saline injection. However, there was no significant difference in the urinary osmolality between wildtype, heterozygous, and Ren-b^Null mice (Fig 4).

Fig 4 — 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.

A separate cohort of Ren-b^Null was placed in metabolic cages before and after 4 weeks on a high salt diet. One animal was excluded due to malocclusion. When compared with parameters at baseline, both WT and Ren-b^Null mice fed a high salt diet for 4 weeks showed an increase in body weight and urine volume and a decrease in food intake and feces excretion (Table 1). Interestingly, Ren-b^Null mice exhibited higher urinary protein excretion compared with wildtype mice at baseline. The level of protein excretion was maintained in Ren-b^Null mice after high salt diet. On the contrary, high salt diet increased urinary protein excretion in WT mice. Since these mice were not implanted with telemeters it is unclear if in this cohort, the urinary protein levels were related to changes in blood pressure. In our previous studies we observed that Ren-b^Null mice fed a normal salt diet exhibit a decreased in plasma renin [30]. However, in Ren-b^Null mice fed a high salt diet we did not observe a statistical difference in renin levels among groups, although a trend toward decrease is evident (p = 0.094). Similarly, we observed a trend toward an increase in heart weight when comparing Ren-b^Null with WT mice on a high salt diet (p = 0.08) (Table 2). No differences in body weight (WT = 28.36±1.16 g vs Ren-b^Null = 29.84±1.45 g), kidney weight, liver weight, spleen weight, and lung weight were found among groups.

Table 1. Male Wildtype (WT) and Ren-b^Null mice were housed in metabolic cages to measure food intake, feces weight, water intake, urine volume, and urinary protein excretion at baseline and after 4 weeks on high salt diet (HSD, 4% NaCl).

	Baseline		HSD		p-value
	WT (n = 5)	Ren-b^Null (n = 5)	WT (n = 5–6)	Ren-b^Null (n = 5–6)	Timepoint	Genotype	Interaction
Body weight (g)	27.26±0.60	26.54±0.75	28.10±0.48	27.60±0.84	0.002	0.533	0.620
Food intake (g/day)	2.63±0.23	2.90±0.33	2.16±0.19	2.10±0.21^*	0.008	0.744	0.383
Water intake (ml/day)	2.99±0.30	3.05±0.34	3.86±0.39	3.24±0.38	0.109	0.511	0.280
Feces weight (g/day)	0.70±0.09	0.85±0.12	0.44±0.03^*	0.49±0.07^*	0.001	0.374	0.488
Urine volume (ml/day)	0.65±0.16	0.61±0.07	1.13±0.12	1.39±0.34	0.015	0.604	0.492
Urinary protein excretion (mg/day)	11.74±5.62	51.66±9.70^#	31.61±7.37	44.12±4.52	0.374	0.009	0.070
Plasma renin (ng/ml)			44.8±12.8	24.3±7.0	0.094

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At the end of the protocol renin was measured from plasma samples. Except plasma renin the data were analyzed by two-way ANOVA with Sidak’s multiple comparisons procedure. Plasma renin data were analyzed by unpaired t-test.

*p<0.05 vs same genotype at baseline and

^#p<0.05 vs WT at same timepoint.

Table 2. Body weight (left column) and tissue weights normalized by body weight (right column) in male Ren-b^Null mice and wildtype littermate controls (WT) fed a high salt diet (HSD, 4% NaCl) for 4 weeks.

	Tissue weight (g)		Tissue weight/body weight (mg/g)
	WT+HSD (n = 6)	Ren-b^Null +HSD (n = 5)	WT+HSD (n = 6)	Ren-b^Null +HSD (n = 5)
Heart	0.125±0.003	0.130±0.003	4.26±0.25	4.72±0.10
Lungs	0.157±0.006	0.150±0.010	5.34±0.17	5.43±0.30
Kidneys	0.177±0.009	0.173±0.006	6.01±0.21	6.27±0.06
Liver	1.217±0.084	1.256±0.066	41.48±2.96	45.55±2.24
Spleen	0.085±0.004	0.080±0.003	2.93±0.25	2.90±0.10

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An unpaired t-test was used for data analysis.

Responses to acute restraint-stress

Since there was no difference in blood pressure in response to the high salt diet, the same animals were further challenged with an acute restraint-stress. After 10 minutes of baseline recording, mice were quickly placed in an acrylic restrainer, and blood pressure and heart rate were recorded for additional 10 minutes. Restraint-stress induced a ~25 mmHg blood pressure elevation in both WT and Ren-b^Null mice (Fig 5A–5C and 5E), which was associated with a robust tachycardic response (Fig 5D and 5F). No difference in the pressor responses to restraint-stress was observed in WT compared with Ren-b^Null mice. Interestingly, Ren-b^Null exhibited elevated heart rate (P_genotype < 0.05), which was more prominent at baseline as it achieved a similar increase compared with WT when exposed to restraint-stress. The individual values of blood pressure and heart rate before and during restraint-stress were plotted separately (Fig 5E and 5F). After the acute stress challenge was completed, the mice were shifted back to low salt diet for 2 weeks. Similarly, restraint-stress induced an increase in both blood pressure and heart rate in both groups, but no difference between WT and Ren-b^Null was observed (Fig 6).

Fig 5 — Acute responses to restraint-induced blood pressure (BP) and tachycardia in male Ren-b^Null 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.

Fig 6 — Acute responses to restraint-induced blood pressure (BP) and tachycardia in male Ren-b^Null 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.

Previous cohorts of Ren-b^Null mice exhibited resistance to body weight gain in response to high fat diet, which was attributed to elevated resting metabolic rate and sympathetic outflow to the brown adipose tissue [23]. Since restraint-stress induces hyperglycemia through the central catecholaminergic neuronal system located in RVLM [31], we evaluated in a separate cohort of male and female mice whether Ren-b^Null mice exhibit differences in the blood glucose response to restraint-stress. Blood glucose levels were measure in 15 minute-intervals before, during, and after mice were restrained. Control mice were kept in their home cages. Restraint-stress induced a significant increase in blood glucose in both wildtype and Ren-b^Null mice (P = 0.0002). However, there was no difference between genotypes (Fig 7A and 7B). Plasma renin and glucocorticoids were measured 30 minutes after mice were released from the restraint. As above, unrestrained Ren-b^Null mice exhibited a trend towards a decrease in plasma renin, but this difference did not reach statistical significance (Fig 7C). No differences in plasma glucocorticoids were observed between animals at the end of the protocol (Fig 7D).

Fig 7 — A) Blood glucose was determined every 15 minutes before, during, and after restraint-stress in Ren-b^Null and wildtype littermates (WT) males and females. A group of unrestrained Ren-b^Null 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-b^Null.

Expression of renin

In models of hypertension such as deoxycorticosterone-acetate-salt model and previous Ren-b^Null cohorts exhibiting higher BP, the elevated BP and sympathetic outflow were associated with overexpression of total renin in different nuclei of the brain including the RVLM. Since the current Ren-b^Null cohorts were normotensive at baseline, we hypothesized that the changes in the phenotype between the previous and the current Ren-b^Null cohorts are attributable to differences in the expression of renin in the brain. Therefore, a new cohort of mice fed a normal sodium diet was used to quantify expression of total renin in the RVLM and the hypothalamus. Contrary to the elevated total renin level observed in the Ren-b^Null mice that exhibit elevated blood pressure previously, we found no increase in the renin mRNA levels in the RVLM in the current cohorts (Fig 8A). Interestingly, there was a significant decrease in renin expression in the hypothalamus (Fig 8B). In addition, given that we have previously shown that Ren-b^Null mice exhibit normal aldosterone levels at baseline but a higher aldosterone secretion when infused with slow-dose Ang II we also measured total renin and renin-a in the adrenal glands. There was no difference in total renin or renin-a among groups (Fig 8C and 8D, respectively) [30].

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-b^Null mice were fed a normal salt diet. The data were analyzed by unpaired t-test. *P<0.05 vs WT.

Discussion

The main finding of this study is that in a mouse model lacking renin-b, there were significant differences in blood pressure among study cohorts which cannot be attributed to changes in dietary sodium intake nor psychological stress. First, we performed retrospective analyses of the blood pressures in five different Ren-b^Null cohorts studied over a 3-year period from 2014 to 2017. Interestingly, we observed the cohorts that exhibited the highest blood pressure were housed and manipulated under conditions that might trigger a stress response. Second, since we have previously observed that ablation of renin-b results in elevated renal sympathetic nerve activity we questioned whether mice lacking renin-b would develop impaired salt handling and thus salt sensitivity. Finally, Ren-b^Null mice on high or low salt diet were further challenged to acute restraint-induced stress. Despite these stimuli, Ren-b^Null mice did not exhibit a clear difference in blood pressure compared with control littermates.

We previously identified that in models of salt-dependent hypertension such as deoxycorticosterone-salt hypertension there is a downregulation of renin-b expression in the brain with a concomitant elevation of the classical renin isoform (renin-a) encoding preprorenin [32]. Similarly, genetic ablation of renin-b resulted in an augmented expression of renin-a in the RVLM, an important node for autonomic control [22]. We reported that disinhibition of the brain RAS due to renin-b genetic deletion results in a mild elevation of blood pressure, which was accompanied by an elevated renal sympathetic nerve activity. Despite the elevated brain RAS activity, Ren-b^Null mice exhibited low plasma renin which can be exacerbated with chronic infusion of Ang II [22,30]. However, after successive rounds of backcross breeding, the Ren-b^Null mice developed a milder phenotype with no apparent blood pressure elevation [30]. Here, we evidenced that these new cohorts of Ren-b^Null mice lacking elevated blood pressure exhibit normal total renin expression in the RVLM, but also a paradoxical downregulation of renin in the hypothalamus. Even though this cannot be compared with the previous cohorts–as renin was not measured in the hypothalamus previously–this observation suggests the lack of elevated renin in the RVLM might contribute to the phenotype variability. Furthermore, these alterations in gene expression might be cell specific. We observed that unlike the previous Ren-b^Null cohorts, there was only a trend toward decrease in plasma renin and no difference in renin expression in the adrenal glands. We considered that environmental or dietary factors might be involved in these phenotypic differences between cohorts.

To test whether ablation of Ren-b results in salt-sensitivity, Renin-b knockout mice and wildtype littermates were first fed a low salt diet for 7 days and subsequently switched to a high salt diet for the next 7 days. A diet high in salt induced a modest increase in blood pressure in both wildtype and renin-b knockout mice. It was recently demonstrated that the C57BL6/J strain is salt-sensitive in response to overactivation of the sympathetic nervous system [33], but this remains controversial as there are other reports indicating that C57BL6/J are salt resistant [34,35]. Contrary to our prediction, renin-b lacking mice did not exhibit an augmented pressor response to salt. In addition to the data shown in the Fig 3, we also noted that there was no difference in systolic blood pressure when the data were separately analyzed during the light and dark cycle (data not shown). Therefore, we concluded renin-b deficiency is insufficient to induce an increased sensitivity to a pressor response to high salt diet.

Several studies indicate that elevated sodium intake can lead to functional and structural cardiac remodeling independent of changes in blood pressure [12,36]. Here, we observed that Ren-b^Null mice exhibit a trend towards increased heart weight when fed a high salt diet, but this did not reach statistical significance. This observation is surprising because we showed that Ang-II infusion induced elevated heart weight in Ren-b^Null mice which was accompanied by induction of pro-inflammatory and pro-fibrotic genes in the myocardium[30]. Studies from Jörg Peters’s laboratory have evidenced that cardiac insult triggers renin-b upregulation in the myocardium [37]. Furthermore, myocardial renin-b overexpression results in cardioprotection against ischemic and hypoglycemic conditions [38,39]. Future studies using conditional and tissue-specific models will be required to elucidate the distinct functions of renin-b in different cells including cardiomyocytes.

Given the lack of the phenotype that is observed in the current cohorts of renin-b knockout mice, we hypothesized that ablation of renin-b leads to an enhanced susceptibility to hypertension-eliciting psychological and physical stressors. Under normal conditions, the activity of the brain RAS is relatively low. This is because it is tonically suppressed by renin-b among other factors. However, it has been shown that animals preconditioned to different stressful conditions can lead to brain RAS disinhibition, a condition that can be recapitulated in the Ren-b^Null model. Importantly, the activation of the brain RAS in response to these environmental factors results in a higher predisposition to hypertension due to enhanced sensitization to a subsequent hypertensive stimulus including Ang-II [40,41]. Therefore, in this current study, we investigated whether renin-b lacking mice are sensitized to an acute restraint-stress induced pressor and metabolic responses. Restraint-stress induced an instantaneous pressor and tachycardiac response in both wildtype and Ren-b^Null mice. However, there was no significant difference in the magnitude of the response and the timing to maximal blood pressure between them. This suggests that ablation of renin-b does not cause a significant susceptibly to this particular acute stressor.

The main limitation of this study is that the animals were exposed to a single acute exposure to a particular stress induced by immobilization. We observed that restraint induces a dramatic response evidenced by a marked and rapid increase in blood pressure and heart rate. Thus, it is possible that the maximal nature of this response may have caused us to not observe an increased response in Ren-b^Null mice to a submaximal stress stimulus. It also remains unclear whether a chronic repetitive daily exposure to not only restraint-stress, but also different types of stressors such as noise, wet cage, and vibrations would unmask the cardiovascular and other phenotypes in Ren-b^Null mice. We recognize that we are limited to recapitulate exactly the environmental conditions that once caused elevated blood pressure in this model, but we are certainly confident that Ren-b^Null mice are resistant to changes in sodium intake and an acute stressor in our current conditions. This indicates that mechanisms unrelated to salt and acute stress alter the cardiovascular phenotype in mice lacking renin-b. We cannot rule out a possibility that a genetic drift causing a downregulation of Ren-a in the central nervous system occurred in our colony after several breeding cycles. Thus, ongoing studies where Ren-b^Null mice are backcrossed to a different genetic background will be useful to determine whether the discrepancies in blood pressure occurred due to spontaneous changes in the DNA along intense inbreeding protocols. Finally, other variables such as the microbiome, variations in temperature and humidity, as well as variations between batches of bedding and chow are difficult to control, but they must be recognized as an important source of variability between experimental cohorts.

Acknowledgments

The authors gratefully acknowledge the technical assistance of Debbie Davis and Kelsey Wackman with animal care. Transgenic mice were first maintained and genotyped at the University of Iowa Gene Editing Facility supported by grants from the National Institutes of Health and the Carver College of Medicine, and then in the Biomedical Resource Center at the Medical College of Wisconsin.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported through research grants from the National Institutes of Health (NIH) to CDS (HL084207 and HL144807), JLG (HL134850), PN (HL153101) and grants from the American Heart Association to CDS (15SFRN23480000), JLGrobe (18EIA33890055). PN was funded with training awards from the NIH for this study (2T32HL007121041, 4T32DK007690, and 5T32HL134643).

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PLoS One. doi: 10.1371/journal.pone.0250807.r001

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26 Apr 2021

PONE-D-21-11323

Studies of Salt and Stress Sensitivity on Arterial Pressure in Renin-b Deficient Mice

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Reviewer #1: Nakagawa et al. come up with a very peculiar concept, i.e., that renin-b suppresses renin-a. What would be the logic of having such a system, and how exactly does this happen? The body has many ways to up- or downregulate renin, including BP, salt and Ang II. Since renin-b remains in the cell, how does it affect renin-a? And then apparently this only applies to cells which generate both renin-a and renin-b? Do we have evidence that both occur in the same cell.

The authors hypothesize that mice lacking renin-b would be more responsive to an acute environmental stressor as consequence of brain RAS disinhibition. In the absence of renin-b, the ‘’disinhibition’’ must already be absent, i.e., renin-a is permanently upregulated? Do they have evidence for this in their various cohorts, i.e., might the different response relate to different upregulations of renin-a? And if so, why do we only see the consequence of this during acute stress? This implies that without stress the upregulated renin-a is still suppressed: by what? What then exactly happens during acute stress (mechanistically)? I fail to understand this.

What about the consequence of renin-b disappearance in the adrenal in the renin-b Null mice? Does it upregulate renin-a at that site? Please provide such data in your model. The renin-b Null mice display proteinuria: why? This occurs in the absence of hypertension. Ren-b Null mice also exhibited an elevated heart rate at baseline, although this is very hard to see in Figure 5. What kind of test was used to reach this conclusion, particularly if it applies only to a few data points?

The authors refer to previous data on renin ‘’showing a trend to decrease’’ after high-salt. I am not sure what this means. At P=0.094 this cannot be stated (nor is it confirmed here), nor does the ‘’trend’’ toward increased heart weight in Table 2. Please refrain from drawing conclusions if P>0.05, and only discuss/show novel data. Figure 7 also shows no difference between WT and renin-b Null mice.

Taken together, after Figure 1, it is very hard to see what exactly is the difference between the WT and renin-b Null mice. It seems there is no difference after all? The authors refer to a ‘’decrease’’ in plasma renin, which is not apparent/significant however. There are no differences in salt sensitivity, and even the stress response does not seem to be different. They mention a trend towards increase in heart weight, but this too is NS. The authors suggest that the phenotype is ‘’milder’’ (in reality it is absent?), and speculate about genetic drift. After reading and re-reading this paper, I fail to see what the message is. It seems that the phenotype is entirely lacking. Preferably, the authors first provide a plausible mechanism by which all of this might occur: how does renin-b affect renin-a.

Reviewer #2: The study Nakagawa et al. starts discussing a controversial blood pressure phenotype found in their previous studies using mice lacking the intracellular renin isoform (Ren-b). The authors suspected the phenotype discrepancy could potentially be related to stressful experimental conditions in previous experiments that would have induced high blood pressure in Ren-b null mice. To test this hypothesis, the impact of low and high salt diets on blood pressure in mice lacking Ren-b was investigated. Additionally, the authors submitted these mice while on low and high salt diets to an acute restraint-stress protocol to evaluate cardiovascular changes. Overall, this study could not show significant cardiovascular changes among Ren-b deleted animals in comparison to controls, but it uses an interesting experimental approach to test cardiovascular physiology in Ren-b null mice. Below are some comments that should help to improve the work.

Major

1) At the end of the abstract the following sentence is written “These studies highlight a complex mechanism that masks/unmasks roles for renin-b in cardiovascular physiology”. What is meant by this sentence, because there was no apparent cardiovascular modulatory effect of Ren-b deletion on high salt diet and stress, neither was proven that stress was correlated to the blood pressure discrepancy in previous studies

2) The authors performed retrospective analyses of the light cycle systolic blood pressure, and argued the blood pressure differences may be related to the room dimensions and people traffic that were not favorable during the recordings of cohorts 1-4. Looking at the original publication it is evident that only Sys pressure is increased only during day time in Ren-b null mice. This information should be included in the present study at least in the text. If light cycle Sys pressure was increased, why this parameter was not investigated separately in the present study rather than the daily average. This should be at least mentioned if there is no difference at baseline as well as during the salt diet challenge. Additionally, do the authors have data recorded during the weekend or holidays, when the room was likely empty, as it seems to be in the dark period? Besides, what is the author’s opinion about the final blood pressure phenotype of Ren-b null mice. Are the data from the previous studies invalid?

3) The authors mentioned that DOCA-salt reduces central Ren-b and increases Ren-a expression, why they did not investigate the mRNA levels in WT brains after high salt diet.

4) The authors wrote the following sentence in the abstract. “When renin-b deficient mice were exposed to a high salt diet for a longer duration (4 weeks), there was a trend for increased myocardial enlargement in Ren-b null mice when compared with control mice”. Indeed, it looks like there was a trend to increased heart weight. However, an age matched group on a normal salt diet should be included to show the initial cardiac weight, to check if this trend was not independent of salt.

5) Why the authors decided to use different time points upon high salt-diet to evaluate BP, stress and morphology? Blood pressure was investigated 1 week after starting the high salt diet, stress 2 weeks and the morphology 4 weeks after.

6) Please give a rational reason why not to investigate the blood pressure response to stress using mice on a normal salt diet too, instead of under low and high salt diets, in particular, because the authors suspected stress could have induced elevation in BP in the cohorts 1-4 in which the animals where likely on a normal salt diet. In the discussion it seems like the experiments were performed in animals on a normal salt diet but this is clearly not the case.

7) In the results it says “Interestingly, an early cohort of Ren-b null mice exhibited higher urinary protein excretion compared with wildtype mice at baseline. The level of protein excretion was maintained in Ren-b null mice after high salt diet. On the contrary, high salt diet increased urinary protein excretion in WT mice.” What is meant by an early cohort of animals? Were those mice hypertensive and, therefore, not from this study?

8) In their discussion the authors mentioned “Similarly, genetic ablation of renin-b resulted in an augmented expression of renin-a in several brain nuclei including the rostral ventrolateral medulla [20]. In the original publication cited, only the RVLM presented significantly increased expression of Ren-a, the other nuclei had a tendency to have increased mRNA. Also, overall brain mRNA level of Ren-a in Ren-b null mice seems not be to altered. Please be more precise when citing data from previous studies in particular those from the own group.

9) Plasma renin activity values shown in figure 7, baseline or after stress, are not higher than 20 ng/mL in average in both WT and Ren-bnull, while animals submitted to a high salt diet in Table 1 WT present 44.8 and Ren-bNull 24.3. However, high salt diet should suppress renin release and peripheral Ang II production, please discuss this surprising result.

10) Plasma renin activity values shown in figure 7, baseline or after stress, are not higher than 20 ng/mL in average in both WT and Ren-bnull, while animals submitted to a high salt diet in Table 1 WT present 44.8 and Ren-bNull 24.3. However, high salt diet should suppress renin release and peripheral Ang II production, please discuss this surprising result.

Minor

1) All Graphs should be marked with A, B, C… on top, otherwise it is confusing to find the graph referred in the text and/or figure legend. Please standardize it, currently some figure legend refer to A,B,C… while others not.

2) Figure 3, top panel: blue dot legend and n are missing.

3) Figure 7 legend has no general description of content as the previous one.

4) On page 3 references should be added to the following sentence “Numerous studies have identified neurogenic actions of elevated salt intake”

5) This sentence in page 3 seems to be incomplete: “For instance, elevated dietary sodium intake sensitizes sympathetic neurons located in the in response to a stimulus [9]”.

6) In the methodology it is written “A cohort of Ren-b Null mice without telemetry implants was placed in metabolic cages before and after 3 weeks on a high salt diet”, and table 1 legend says “Table 1: Male Wildtype (WT) and Ren-b null mice were housed in metabolic cages to measure food intake, feces weight, water intake, urine volume, and urinary protein excretion at baseline and after 4 weeks on high salt diet (HSD, 4% NaCl)”. What is correct, 3 or 4 weeks after HSD?

7) Table 2 – The legend says “Body weight (left column) and tissue weights normalized by body weight (right column)”. The body weight is not shown in this table. The left column shows tissue organ weights, correct?

8) Methodology of Experiment 2 – please provide more information regarding this experiment, particularly the exact tonicity of the injected solutions, the time course of the experiment and how the urine was sampled.

Reviewer #3: The authors compared the salt- and stress- sensitivity of renin-b null and wild type mice with respect to blood pressure, heart rate and renal and metabolic parameters. Their hypothesis was, that under salt- or restrain-stress renin-b null mice may have elevated blood pressure and/or heart rates.

The rational to perform the present study was as follows: The authors previous showed that the genetic removal of renin-b triggered a mild increase in blood pressure and sympathetic nerve activity. The authors previously hypothesised that renin-b maintains brain RAS activity at normal physiological levels by suppressing the classical renin isoform and thus, renin-b deletion would des-inhibit a brain RAS. However, in later studies with other cohorts of renin-b null and wild type mice differences in blood pressure or heart rate were not observed any more. The authors hoped to attribute these discrepancies to differences responses to salt intake and/or stress.

The experiments were very well designed and performed. From the methodological point of view, I have only minor criticism (see below).

However, with respect to the original hypothesis (renin-b inhibits renin-a expression in the brain and des-inhibition of renin-a in the absence of renin-b increases blood pressure) it would be essential to measure renin-a expression again in the brain (or in specific brain areas), i. e. to demonstrate that renin- a mRNA it is still elevated (or not). Only thus the original hypothesis can be supported or must be discarded.

Minor comments:

I would not rely on „trends”( heart weight/BW; plasma renin levels) – moreover, a p-value around 0,09 may not even be considered as a trend.

Basal urinary protein excretion were significantly higher in renin-b null – with no further increase under HSD. Please explain possible reasons.

Table 2: please add p-values

Heart rate Fig. 5: the basal heart rates were initially not different (minutes -10 to -5). This was followed by a transient increase, but just before restrain-stress there was again no difference when compared to wild type.

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Reviewer #3: Yes: Joerg Peters

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PLoS One. 2021 Jul 28;16(7):e0250807. doi: 10.1371/journal.pone.0250807.r002

Author response to Decision Letter 0

21 Jun 2021

Please, see our responses to the reviewers and the editor in the attached document.

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PLoS One. doi: 10.1371/journal.pone.0250807.r003

Decision Letter 1

Michael Bader

7 Jul 2021

PONE-D-21-11323R1

Studies of Salt and Stress Sensitivity on Arterial Pressure in Renin-b Deficient Mice

PLOS ONE

Dear Dr. Nakagawa,

Please submit your revised manuscript by Aug 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: All questions have been addressed, and the authors provided novel data on renin a expression in the adrenal. Please add these to the MS, and discuss them accordingly. These are highly relevant, as their concept is that renin b somehow suppresses renin a. Apparently, this is not true in the adrenal? Why would this only happen in the brain?

Reviewer #2: (No Response)

Reviewer #3: My comments have been addressed. The lack of change in renin (renin-a) expression fits to the lack of change in blood pressure. However, there was a decrease - not increase of renin (a) in the hypothalamus. This contradicts the authors conclusion that renin-b decreases renin-a expression. Furthermore, if renin-a in the hypothalamus has any role at all, this decrease should have some consequences. This need to be stated and discussed.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2021 Jul 28;16(7):e0250807. doi: 10.1371/journal.pone.0250807.r004

Author response to Decision Letter 1

12 Jul 2021

We carefully addressed the reviewers' inquiries. Our responses are attached in a separate word document.

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PLoS One. doi: 10.1371/journal.pone.0250807.r005

Decision Letter 2

Michael Bader

15 Jul 2021

Studies of Salt and Stress Sensitivity on Arterial Pressure in Renin-b Deficient Mice

PONE-D-21-11323R2

Dear Dr. Nakagawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0250807.r006

Acceptance letter

Michael Bader

19 Jul 2021

PONE-D-21-11323R2

Studies of Salt and Stress Sensitivity on Arterial Pressure in Renin-b Deficient Mice

Dear Dr. Nakagawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Data Availability Statement

All relevant data are within the manuscript.

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PERMALINK

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

Pablo Nakagawa

Javier Gomez

Ko-Ting Lu

Justin L Grobe

Curt D Sigmund

Roles

Abstract

Introduction

Methods

Animals

Blood pressure measurements

Assessment of salt sensitivity

Experiment 1

Fig 1. Experimental protocol.

Experiment 2

Experiment 3

Responses to restraint stress in mice fed high or low salt diets

Experiment 4

Plasma renin measurements

Expression of renin in the central nervous system and adrenal glands

Experiment 5

Ren1

Renin-a

18s

Statistical analysis

Results

Retrospective analysis of the arterial blood pressure in Ren-bNull cohorts

Fig 2. Cohort analysis of arterial blood pressure.

Effect of high salt diet on blood pressure in Ren-bNull mice

Fig 3. Blood pressure response to salt.

Fig 4. Renal function.

Table 1. Male Wildtype (WT) and Ren-bNull mice were housed in metabolic cages to measure food intake, feces weight, water intake, urine volume, and urinary protein excretion at baseline and after 4 weeks on high salt diet (HSD, 4% NaCl).

Table 2. Body weight (left column) and tissue weights normalized by body weight (right column) in male Ren-bNull mice and wildtype littermate controls (WT) fed a high salt diet (HSD, 4% NaCl) for 4 weeks.

Responses to acute restraint-stress

Fig 5. Blood pressure response to restraint stress (HSD).

Fig 6. Blood pressure response to restraint stress (LSD).

Fig 7.

Expression of renin

Fig 8.

Discussion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Michael Bader

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael Bader

Roles

Author response to Decision Letter 1

Decision Letter 2

Michael Bader

Roles

Acceptance letter

Michael Bader

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Retrospective analysis of the arterial blood pressure in Ren-b^Null cohorts

Effect of high salt diet on blood pressure in Ren-b^Null mice

Table 1. Male Wildtype (WT) and Ren-b^Null mice were housed in metabolic cages to measure food intake, feces weight, water intake, urine volume, and urinary protein excretion at baseline and after 4 weeks on high salt diet (HSD, 4% NaCl).

Table 2. Body weight (left column) and tissue weights normalized by body weight (right column) in male Ren-b^Null mice and wildtype littermate controls (WT) fed a high salt diet (HSD, 4% NaCl) for 4 weeks.