bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracerebroventricular injection of liposome‐entrapped GABA attenuates the renal sympathetic nerve activity response evoked by central administration of bicuculline in anesthetized rats
Authors: Vaz Gisele Cristiane , Bahia Ana Paula Oliveira , Santos Robson Augusto Souza , Frézard Fréderic , Fontes Marco Antônio Peliky
Published: June 2021

Liposomes are lipid vesicles that allow a sustained release of entrapped substances. GABA is the most prevalent inhibitory neurotransmitter of the central nervous system. In this study we tested the efficacy of liposome‐entrapped GABA (GL) in attenuating the cardiovascular response evoked by intracerebroventricular (icv) administration of GABAA antagonist bicuculline methiodide (BMI). Liposomes were prepared by the freeze‐thawing method followed by extrusion (200‐nm final vesicle diameter). Under tribromoethanol anesthesia (250 mg/Kg i.p.), guide cannula was implanted into the lateral ventricle of Wistar rats. In two groups of animals (n=6–8), GL or empty liposomes (EL, 0.9% NaCl) were injected icv 48 hours before the GABAA antagonist (BMI, 0.5 nmol/2μL). The icv injection of BMI produced similar increases in HR in both groups (EL: 49 ± 15 and GL: 30 ± 10 bpm). Minor changes in blood pressure were observed. However, the evoked increase in renal sympathetic nerve activity showed in EL group was greatly attenuated in GL group (43 ± 8 vs. 12 ± 9 %, P=0.034). Current data extends our previous findings suggesting that liposome‐entrapped GABA is a promising tool for long‐term intervention in central nervous system. Support: CNPq/FAPEMIG.
Menstrual cycle and sympathetic neural activity in humans: A retrospective study
Authors: Carter Jason R , Fu Qi , Minson Christopher T , Joyner Michael J
Published: June 2021

Some studies report an increase of resting muscle sympathetic nerve activity (MSNA) during the midluteal (ML) phase of the menstrual cycle when compared to the early follicular phase (EF), while others do not. The inconsistent findings may be due, in part, to variable surges in estradiol and progesterone. We tested the hypothesis that the degree of sympathoexcitation during the ML phase (ΔMSNA) is associated with changes in estradiol (ΔE2) and progesterone (ΔP). Retrospective analysis of previous studies with recordings of mean arterial pressure (MAP), MSNA, E2, and P during both EF and ML phases yielded 30 eumenorrheic women (age, 18–51 yrs). ML phase increased E2 (37±2 to 117±9 pg/ml; p<.001), P (1±0 to 11±1 ng/ml; p<.001), and MSNA (12±1 to 15±1 bursts/min; p=.02), but did not alter MAP (83±2 to 83±2 mmHg; p=.91). ΔMSNA was correlated to ΔE2 (r=−.50, p=.005) and ΔE2/ΔP (r=−.52, p=.004), but not ΔP (r=.21, p=.26). There was no association between ΔMAP and ΔE2 (r=−.13, p=.49), ΔP (r=−.04, p=.83), or ΔE2/ΔP (r<.01, p=.98). In conclusion, sympathoexcitatory responses to the ML phase appear to be associated with the degree of sex steroid surges. While E2 had a stronger influence on MSNA than P, it is worthy to note that a strong association with ΔMSNA persisted when both ΔE2 and ΔP were considered. This dynamic interaction between E2, P, and MSNA likely explains previously reported inconsistencies in the field. Supported by NIH.
Ubiquitination is involved in the alteration of peripheral acetylcholine mediation in the aging heart
Authors: Freeling Jessica Lynn , Li Yifan
Published: June 2021

It is known that aging and heart disease both result in an increase in sympathetic nervous system activity and a decrease in parasympathetic nervous system (PSNS) activity, which may contribute to age‐related cardiac dysfunction and remodeling. However, the mechanisms underlying impairment of the PSNS in aging remain to be fully understood. Utilizing young (1–2 month) and old (11–12 month) mice, it was determined that a peripheral and pre‐synaptic mechanism is involved in the diminishment of baroreflex sensitivity (BRS) and loss of response to rostral‐severed vagal stimulation within the hearts of aging mice. Analysis of protein expression of aging mouse atria with fat pad reveals alteration of the proteins involved in acetylcholine (Ach) mediation. A significant decrease in Ach synthesizing protein, choline acetyltransferase (ChAT), as well as a significant decrease in the upper band of the Ach packaging protein, vesicular Ach transporter (VAchT) was found. In contrast, a significant upregulation of the upper band of the rate‐limiting protein, choline transporter (CHT), is seen in the aging animals indicative of a compensatory mechanism. Further investigation utilizing immunoprecipitation has revealed that the upper band of VAchT, but not CHT is ubiquitinated. Given the role of ubiquitination in protein trafficking and turnover, the altered ubiquitination of VAChT seen in this study may impact VAChT distribution and activity, and thus PSNS transmission at intra‐atrial ganglia in aging. This work was supported by AHA grant No.0835256N, NIH INBRE grant No.P20 RR016479, NIH grant No. 1R03AG033291, and the BBS bridge grant.
Central angiotensin II induces sympathoexcitation and attenuates the open loop baroreflex gain without altering central baroreflex characteristics
Authors: Onitsuka Ken , Hosokawa Kazuya , Sakamoto Takafumi , Sakamoto Kazuo , Tobushi Tomoyuki , Fujino Takeo , Saku Keita , Matsukawa Ryuichi , Hirooka Yoshitaka , Sunagawa Kenji
Published: June 2021

Background

Central angiotensin II (ANG II) induces sympathoexcitation and hypertension. However, how ANG II impacts on the open loop baroreflex (BR) characteristics and thereby stabilization function of arterial pressure (AP) remains unknown.

Methods

In 7 anesthetized rats, we examined the acute effects of intracerebroventricular infusion of ANG II (2 ng/kg/min) on the open loop BR characteristics. We changed carotid sinus pressure (CSP) stepwise from 60 to 170 mmHg, while measuring AP and splanchnic sympathetic nerve activity (SNA). We characterized the central, peripheral and total BR loops by the CSP‐SNA relation, the SNA‐AP relation and the CSP‐AP relation, respectively.

Results

In the central loop, ANG II markedly increased SNA (ΔSNA=57.2±17.7%, p<0.01) irrespective of CSP without attenuating the maximum gain (1.99±0.25 vs. 3.58±1.14 %/mmHg, ns) or response range (70.8±5.0 vs. 115.0±27.0%, ns). In the peripheral loop, although ANG II markedly increased AP (ΔAP=49.8±10.4 mmHg, p<0.01), the AP response plateaued in the high SNA range. In the total loop, the plateaued AP response decreased the maximum gain (1.93±0.15 vs. 0.629±0.162, p<0.01) and narrowed the response range (66.9±4.7 vs. 39.0±8.8 mmHg, p<0.01).

Conclusion

Central ANG II induces sympathetically mediated hypertension and impairs AP stabilization function. This study was supported by the research grants from MEXT and MHLW, Japan.
Augmented alpha‐adrenergic vasoconstriction during exercise in human metabolic syndrome
Authors: Limberg Jacqueline K , McKenna Edward J , Sebranek Joshua J , Walker Benjamin J , Hagen Scott A , Morgan Barbara J , Schrage William G
Published: June 2021

The contribution of α‐adrenergic vasoconstriction during exercise in animal models of metabolic syndrome (MS) is controversial and the relevance to human MS is unclear. We hypothesized humans with MS (n=11) would exhibit greater α‐adrenergic vasoconstriction during exercise compared with healthy controls (n=16) and greater constriction would correlate with higher muscle sympathetic nerve activity (mSNA). We measured forearm blood flow (BF, Doppler ultrasound) and calculated changes in conductance (BF/blood pressure) with local intra‐arterial infusion of adrenergic agonists [phenylephrine (PE, α1), clonidine (CL, α2)] during dynamic forearm exercise (15% maximal effort). mSNA was measured in a subset of subjects (n=15, microneurography of the peroneal nerve). mSNA was higher in MS (66±9 vs 35±4 bursts/100 heart beats, p<0.01) and did not change with exercise. Infusion of PE decreased conductance similarly between groups. In contrast, MS subjects exhibited greater constriction to CL infusion compared with controls (p<0.01). Greater CL‐mediated constriction was associated with higher mSNA (r=0.51, p=0.05). These findings demonstrate greater α2‐adrenergic vasoconstriction during exercise in human MS which is related to higher mSNA. Such relationships may lead to impaired blood flow distribution and blood pressure regulation during exercise in MS. Support: AHA 10PRE3870000, NIH HL091397.
Acute phosphodiesterase inhibition improves functional muscle ischemia in patients with Becker muscular dystrophy
Authors: Martin Elizabeth Anne , Walker Ashley E , Scott Bryan L , Malott Teresa C , Singh Nirmal , Gurudevan Swaminatha V , Johannes Jimmy , Elashoff Robert M , Thomas Gail D , Victor Ronald G
Published: June 2021

Loss of sarcolemmal nitric oxide synthase (nNOS) engenders ischemia of exercising dystrophin‐deficient muscles of mdx mice and boys with Duchenne muscular dystrophy. We tested if muscle ischemia also occurs in Becker muscular dystrophy (BMD), a milder disease often caused by dystrophin mutations involving the nNOS binding site, and is improved by tadalafil, a phosphodiesterase (PDE5A) inhibitor that enhances cGMP/NO signaling. We measured reflex vasoconstriction (decreased forearm muscle oxygenation [ΔHb02, near infrared spectroscopy] during lower body negative pressure [LBNP]) at rest and during rhythmic handgrip (HG) in 5 male controls (Ctrls) and 10 men with BMD who underwent a placebo‐controlled cross‐over trial of single‐dose (20 mg) tadalafil. At baseline, HG greatly attenuated vasoconstriction in Ctrls (ΔHb02:−393±89 vs. −91±40 units, p<.01; rest vs. HG) but caused no attenuation in BMD (−381±45 vs. − 374±46). Tadalafil markedly improved ischemia in BMD (ΔHb02:− 439±70 vs. −230±54, rest vs. HG; p=0.014) whereas placebo had no effect. These data provide the first evidence in man that PDE5A inhibition can improve blood flow regulation in dystrophin‐deficient skeletal muscle. Funded by MDA 201149.
Evidence for GPR107 as a cognate receptor for neuronostatin
Authors: Yosten Gina L. C. , Samson Willis K.
Published: June 2021

Neuronostatin is a recently described peptide hormone that is derived from the somatostatin preprohormone. We previously have shown that neuronostatin administered into the lateral cerebroventricle of adult, male rats led to a significant increase in mean arterial pressure (MAP). We sought to determine if this effect of neuronostatin was physiologically relevant; however, any attempt to disrupt the production of neuronosatin would compromise somatostatin as well. We therefore decided to assess instead the effect of loss of the neuronostatin receptor on cardiovascular function. To identify the receptor, we first screened four neuronostatin‐responsive cell types for the expression of orphan GPCRs, and found that GPR107 was present in all cell types tested. We next knocked down the expression of GPR107 in vitro using siRNA, and found that loss of GPR107 abolished neuronostatin‐induced cFos expression. Loss of endogenous GPR107 in male rats ablated the elevation in MAP caused by neuronostatin treatment, and blunted the cardiovascular responses of animals in a baroreceptor sensitivity test. These data suggest that GPR107 is a cognate receptor for neuronostatin, and that this hormone and its receptor are physiologically relevant regulators of cardiovascular function. Supported by NIH#HL66023.
mTOR/S6K Signaling: A Novel Effector of Neuronal Action of Angiotensin II
Authors: Muta Kenjiro , Grobe Justin L. , Sigmund Curt D. , Rahmouni Kamal
Published: June 2021

The renin‐angiotensin system (RAS) is critically involved in arterial pressure regulation. Angiotensin II (Ang II), the main effector of the RAS, acts in several brain nuclei including the subfornical organ (SFO) to increase arterial pressure by stimulating Ang II type 1 receptors (AT1R). However, the signaling mechanisms of neuronal AT1R remain poorly defined. Neuronal mammalian target of rapamycin (mTOR) and its downstream effector, p70S6 kinase (S6K), have emerged as important regulators of arterial pressure. Thus, we hypothesized that mTOR/S6K signaling mediates brain Ang II actions. In hypothalamic neuronal cell line (GT1‐7), transfected with AT1aR, Ang II (100 nM) caused a time‐dependent increase in S6K activity with a maximum effect at 15 min (3.4‐fold; P<0.05 vs. vehicle). Pre‐treatment with AT1R blocker (1 μM losartan) completely abolished Ang II‐induced activation of S6K. We also examined, by immunohistochemistry, the level of phospho‐S6 (a readout of S6K activity) in the brain of transgenic sRA mice which have overactive brain RAS. The SFO of female sRA mice exhibited significantly (P=0.02) elevated phospho‐S6 positive cells (64±6) relative to littermate controls (36±8). This increase was specific to the SFO as there was no difference in phospho‐S6 levels in other forebrain nuclei in sRA vs. control mice. This study has identified mTOR/S6K as a novel mechanism of AT1R signaling in neurons.
A novel model of conditionally inducible angiotensin production in the brain: investigations of sodium and fluid intake
Authors: Coble Jeffrey Paul , Grobe Justin , Cassell Martin , Sigmund Curt
Published: June 2021

Transgenic mice expressing human renin via the neuron‐specific synapsin promoter (sR) were bred with ubiquitous CAG promoter, conditionally inducible human angiotensinogen (AGT) mice (ARed). Recombination of the ARed transgene by Cre‐recombinase results in loss of a dsRed marker and gain of AGT expression. Adenovirus encoding Cre (AdCre) was injected into the lateral cerebral ventricle (ICV) of sRARed and littermate control mice. At baseline, total fluid and sodium intakes of sRARed mice (0.30±0.02 mL/day; 0.97±0.15 mEq/day, n=12) was similar to littermates (0.24±0.03 mL/day; 0.63±0.03 mEq/day, n=16) in a two‐bottle choice paradigm (water vs 0.15 M NaCl). Three weeks after AdCre injection, sRARed mice increased both total fluid and sodium intakes in comparison to baseline (0.46±0.05 mL/day, P=0.01; 1.79±0.20 mEq/day, P<0.01, n=12) and littermate controls (0.27±0.04 mL/day, P<0.01; 1.03±0.11 mEq/day, P<0.01, n=16). Mice expressing a reporter construct sensitive to Cre‐recombination (ROSA‐tdTomato) were injected using the same methodology as the sRARed mice. ICV delivery of AdCre into ROSA‐tdTomato mice resulted in reporter gene expression throughout the ependyma but also within the subfornical organ and median preoptic nucleus. We developed a model of conditionally inducible expression of AGT, which can be used to temporally and spatially control the production of angiotensin peptides in the brain.
Age dependent cardiovascular responses to angiotensin II (ANG II) in conscious mice
Authors: Qi Wei , Gera El-Sayed SB , Smith Francine G
Published: June 2021

ANG II plays a critical role in regulating cardiovascular homeostasis. By binding to type 1 receptors, ANG II increases vascular resistance and thereby arterial pressure. The expression of type II receptors, which promote vasodilatation, is upregulated in the post natal period and may result in different cardiovascular responses to ANG II early in life. This study was designed to evaluate cardiovascular responses to ANG II in two groups of conscious mice (Group I: 4 weeks, n=5; Group II: 10–14 weeks, n=8). Experiments consisted of measurements of mean arterial pressure (MAP) and heart rate (HR) for 20 min before (Control, C) and 30 min after I.V. infusion of one of seven doses of ANG II (0–10 ng/g/min). ANG II was associated with a dose‐dependent increase in MAP in both groups reaching a maximum at 5.0 ng/g/min (EC100) in group I (C 99±11; Max 126±16 mmHg) and 0.5 ng/g/min (EC100) in group II (C 113±9, Max 139±22 mmHg). The time to peak was longer and the duration of the MAP response was less in group I as compared to group II. In group I, in response to EC100, HR was decreased from 563±103 (C) to 467±62 bpm, yet remained unchanged in group II from 503±87 bpm (C). These results provide evidence that the cardiovascular responses to acute ANG II infusion in young mice are developmentally regulated. Mechanisms underlying these age‐dependent differences are not yet clear but could reflect developmental regulation of ANG II receptors.
AT1 receptor antagonism but not mineralocorticoid receptor blockade lowers blood pressure in obese Zucker rats
Authors: do Carmo Jussara M , da Silva Alexandre A , Hall John E
Published: June 2021

Obesity is associated with mild activation of most of components of the renin‐angiotensin‐aldosterone system. Chronic angiotensin II type 1 (AT1) receptor antagonism with losartan markedly lowered blood pressure (BP) in obese Zucker rats. However, the importance of mineralocorticoid receptor (MR) activation in contributing to the increased BP in obese Zucker rats is still unknown. In this study we tested the long‐term cardiovascular effects of the MR antagonist spironolactone alone or in combination with losartan in lean (n=7) and obese (n=5) Zucker rats. The rats were implanted with telemetry pressure transmitters for determination of BP and heart rate (HR) 24‐hrs/day. After a 5‐ day control period, spironolactone (40 mg/kg/day, PO twice daily) was given for 10 days followed by a 10‐day combined treatment with losartan (20 mg/kg/day, PO). Compared to lean rats, obese rats were hypertensive (115±4 vs 104±2 mmHg) and had lower HR (355±9 vs 393±7 bpm). Chronic MR blockade alone did not alter BP or HR in lean (104±2 to 101±2 mmHg and 393±7 to 387±7 bpm) or obese (115±4 to 118±4 mmHg and 355±9 to 353±12 bpm) rats. Combined treatment reduced BP in obese and lean rats by 31±3 and 21±1 mmHg, respectively, while increasing HR only in obese rats (18±3 bpm). These results suggest that the increased BP observed in obese Zucker rats is dependent on AT1 receptor activation but not on MR activation. (NHLBI‐PO1HL51971/AHA SDG5680016)
Losartan attenuates cardiac remodeling but does not prevent vascular dysfunction in isoproterenol‐treated rats
Authors: Davel Ana Paula , Victorio Jamaira Aparecida , Clerici Stefano Piatto , Rossoni Luciana Venturini
Published: June 2021

Overactivation of β‐adrenoceptors (β‐AR) induced by isoproterenol (Iso) results in cardiac remodeling and vascular dysfunction. We tested the hypothesis that angiotensin II (AII) via AT1 receptor could be involved in the cardiovascular effects of Iso treatment. Rats were treated with Iso (0.3 mg/kg/day, s.c.) or vehicle and co‐treated or not with the AT1 receptor antagonist losartan (Los, 40 mg/kg/day, p.o.) for one week. Mean arterial pressure and heart rate were not changed by treatments. Iso enhanced right and left ventricular mass and Los attenuated this effect. In aortas, the relaxation responses to acetylcholine and sodium nitroprusside were similar among groups. Iso increased the contractile response of aorta to phenylephrine (Phe) and Los did not alter this effect. Incubation with the inhibitor of nitric oxide (NO) synthase L‐NAME abolished differences in Phe contraction among groups. Data suggest that blockade of AT1 receptor with Los partially prevents the cardiac remodeling induced by Iso, but not the enhanced contractility of aorta related to a reduced NO modulation. Thus, AII via AT1 receptor seems do not mediate the vascular effects of β‐AR overactivity.

Financial support: FAPESP/Brazil
ageLOC Vitality enhances sexual functions in normal and impotent models
Authors: Wu Zimei , Dong Yunzi , Zhao Chunsheng , Zhang Yan , Tan Ningzhi , Lu Jihong , Zhu Jia-Shi
Published: June 2021

Based on the differentiated gene expression in skeletal muscle and brain tissue between the young (5 mo) and old (25 mo) mice, ageLOC Vitality was formulated with ingredients (Cordyceps sinensis Cs‐4 and extracts from ginseng and pomegranate) that oppose the age associated changes. Previously we reported that it improved maximal/endurance exercise, glucose metabolism, anti‐oxidation and mitochondria functions in old mice (FASEB J. 2011 25:1114.9; 1049.2). In this study we examined its effect on sexual functions in rats and mice. (1) Rats (3 mo) underwent orchiectomy (OE), resulting in prolongation of erection latency (EL) (p<0.01). After >5 wks of rest, OE rats received 3 days of vehicle or Vitality at 0.2 or 0.8 g/kg by gavage. Vitality shortened EL in OE rats that received a single dosing at both dosages of Vitality and after 3 days at 0.8 g/kg, compared to OE controls (p<0.01). (2) ICR mice (8 mo) received Vitality at 0.4 or 0.8 g/kg for 20 days and were examined for capture latency (CL) and capture times within 20 min (CT20). There were no significant changes in plasma Dihydrotestosterone or Follicle‐Stimulating Hormone on Day 20, compared to controls. CL was shortened with Vitality 0.4 g/kg (p=0.03). CT20 was increased by 78% on Day 3 at 0.4 g/kg (p<0.01), and 109% on Day 20 at 0.8 g/kg (p<0.01). In conclusion: ageLOC Vitality enhances sexual behavior by shortening EL in OE rats, and by shortening CL and increasing CT20 in mice.
Enhanced uterine contractility in an animal model of endometriosis
Authors: Hernandez Siomara , Cruz Myrella L , Ramirez Antonio , Torres-Reveron Annelyn , Appleyard Caroline B
Published: June 2021

The uterus is composed of smooth muscle layers allowing its regulation. Endometriosis is defined as the presence of endometrium‐like tissue outside the uterus and is characterized by peritoneal inflammation, cysts, adhesions, infertility and chronic pelvic pain.

Aim

Investigate the impact of endometriosis on uterine muscle motility.

Methods

Endometriosis was induced in female Sprague‐Dawley rats by suturing uterine horn tissue next to the intestinal mesentery (Endo). Sham rats had sutures only. All animals were sacrificed in estrous phase and examined for cysts. Tissues were analyzed for macroscopic and microscopic damage, myeloperoxidase (MPO) levels, and mast cell numbers. Uterine strips were mounted in organ baths to study in vitro isometric contractile responses of longitudinal and circular muscle to carbachol (10−8–10−3M).

Results

Only Endo rats developed cysts. These animals had increased tension in uterine longitudinal muscle, more colonic damage (macroscopic 1.61 ± 0.26 vs. 1.13 ± 0.57, microscopic 4.89 ± 0.46 vs. 3.55 ± 0.45; p=0.056), more mast cells in the uterus (p<0.05), and higher MPO levels (1.5‐fold and 2‐fold for colon and uterus respectively) than Sham (n = 9 ± s.e.m.).

Conclusions

Our data indicate that the pathophysiological processes associated with this animal model can influence evoked uterine contractility. R15AT006373, R25GM082406, HPD 335731 & CFRDG 335513 from NSU
Male‐dominant up‐regulation of organic anion transporter 1 (Oat1) and 3 (Oat3)
Authors: Henjakovic Maja , Wegner Waja , Burckhardt Birgitta C. , Burckhardt Gerhard
Published: June 2021

Male‐dominant and testosterone‐dependent expression of Oat1 and Oat3, which are involved in the transport of organic anions across the cell membrane, have been shown in renal proximal tubular cells. The aim of this study was to identify the transcriptional regulators of sex‐dependent Oat1 and Oat3 expression. The activities of Oat1 and Oat3 promoter constructs were investigated using luciferase assays. Expression profiling was done using SurePrint G3 rat GE 8×60K microarray. RNA was isolated from renal cortical slices of four adult rats per gender. The genes which are expressed in the proximal tubular cells were filtered using a transcriptome database. Our results revealed that predicted androgen responsive elements in the promoter regions of Oat1 and Oat3 are not functional, and that both transporters are not directly activated by the classical androgen receptor mediated transcriptional pathway. Microarray screening revealed as potential transcriptional regulators amongst other, male‐dominant genes B‐cell lymphoma 6 (Bcl6), polymerase (RNA) III (DNA‐directed) polypeptide G (32kD) and hydroxysteroid (17‐beta) dehydrogenase 1. The transcription factor Bcl6 activated the promoters of Oat1 and Oat3. The renal male‐predominant expression of Oat1 and Oat3 appears to be directly regulated by transcription factor Bcl6 and not by classical androgen receptor mediated pathway. Supported by: DFG‐Bu571/8‐1
Estrogen supplementation in female mice yields higher sodium (Na) retention than that of testosterone in male mice
Authors: Rouch Al , Curtis Kath , Fan Liming , Kudo Lucia , Toal Sheri , Naukam Rebecca
Published: June 2021

Our purpose was to determine the effects of estrogen and testosterone supplementation on Na retention, blood pressure (MAP), and mRNA levels of renal Na transporters in mice. Gonadectomized female and male mice (CD1, 30–35g, 4–6 weeks old) received a pellet of 1.5 mg 17β‐estradiol (E2) and 5 mg testosterone, respectively. In metabolic cages mice consumed 1% salt diet for 10 days, 4% salt for the next 10 days, and 1% salt for the final 10 days. Na intake (Nai) and renal Na excretion (Nae) were measured daily. MAP was measured in the final 4–5 days of each period via tail‐cuff. Real‐time PCR was conducted on kidney tissue processed on the final day of each period. Nae/Nai % (mean ± se):

(n=4)
1% Salt
4% Salt
1% Salt

NF
57.3 ± 3.9
59.9 ± 2.5
54.0 ± 3.3

NM
47.9 ± 3.8
55.8 ± 2.9
48.4 ± 2.9

OE
28.3 ± 1.3*
32.4 ± 1.7*
37.8 ± 2.6*#

CT
53.5 ± 3.1
56.7 ± 3.5
47.6 ± 3.3

NF & NM: normal female and male mice, OE: ovariectomized + E2, CT: castrated + T,

‐ different from all other values (p<0.001);

‐ different from first 1% OE period (p<0.05)

MAP (mmHg) of OE mice were 95.7 ± 4.1, 79.5 ± 4.6, 93.0 ± 4.9 (1%, 4%, 1%, respectively) and not different from those of NF. PCR data showed E2‐induced upregulation of NCC, NKCC2, and ENaC mRNA.

Conclusion

E2 supplementation in female mice produced higher Na retention than that of T in male mice without enhancing MAP. Study supported by OCAST (OK Center for the Advancement of Science and Technology) # HR 09‐123, and OK Epscor INBRE #P20RR016478.
Mechanism of Protection of the Aldosterone Signaling Pathway in the Rat Collecting Duct during Pregnancy
Authors: West Crystal , Masilamani Shyama ME
Published: June 2021

Recently, we found that renal epithelial sodium channel (ENaC) protein and activity are increased in the late pregnant (LP) rat, via a mineralocorticoid receptor (MR)‐mediated mechanism. The aim of this study is to examine the mechanism of protection of MR‐mediated ENaC stimulation in pregnancy when circulating cortisol and progesterone are also increased, two factors known to interfere with MR‐mediated effects. We measured serum and renal levels of aldosterone and progesterone using RIA assays in virgin (V, n=14) and LP (n=8) rats. Further, we determined whole kidney protein abundance of the progesterone receptor (PR) and of the enzyme 11β HSD2 which metabolizes cortisol. Plasma aldosterone (V= 0.82±0.14; LP=2.31±0.32 nmol/L, p<0.05) and renal aldosterone (V=0.82 ± 0.06; LP=1.62 ± 0.22, nmol/L/g kidney), p<0.05) were increased with pregnancy. Interestingly, plasma progesterone increased (V=102 ± 11; LP=182 ± 9, nmol/L), p<0.05) while renal progesterone did not change (V=358 ± 61; LP=339 ± 66, nmol/L/g kidney, NS). Although PR abundance was unchanged (LP= 94±6 % of virgin n=7, NS), we found an increase in 11βHSD2 abundance (LP=135±5 % of virgin n=7, p<0.05). Thus renal MR‐mediated effects are protected in pregnancy in part by 1) increasing cortisol metabolism via increased 11βHSD and 2) preventing competitive inhibition of the MR by renal adaptations that prevent an increase in local progesterone levels.
Pregnancy amplifies the deleterious effects of nanoparticle exposure on microvascular nitric oxide: a role for oxidant stress
Authors: Boegehold Matthew A , Nurkiewicz Timothy , Masilamani Shyama ME
Published: June 2021

Exposure to environmental nanoparticles (NP) may pose a significant risk to cardiovascular health. In male rats, NP inhalation reduces systemic arteriolar endothelium‐dependent dilation due to scavenging of nitric oxide (NO) by reactive oxygen species (ROS). Since NO plays a key role in microvascular adaptations to pregnancy, we assessed the effect of pulmonary NP exposure on oxidant levels and NO‐dependent dilation in extrapulmonary arterioles of pregnant rats. Virgin (V) and mid‐pregnant (MP, Day 11–12) rats were exposed to TiO2 NP via intratracheal instillation (500 μg deposition). After 24 hours, spinotrapezius muscle arterioles were probed for ROS via ethidium bromide (EB) fluorescence or challenged with ACh before vs. after treatment with L‐NMMA, a NOS inhibitor. TiO2 exposure significantly increased arteriolar EB fluorescence in both groups, but this effect was greater in MP (grayscale units: shams =143±3, NP =172±6) than in V (shams= 160±3, NP =176±5). TiO2 also reduced the L‐NMMA‐sensitive component of ACh‐induced dilation, but to a greater extent in MP. These results suggest that the increase in arteriolar ROS and decrease in NO linked to NP exposure is exacerbated in pregnancy. Since NO contributes importantly to systemic vasodilation in pregnancy, this effect of NP could compromise blood pressure regulation in this setting. AHA 09GRNT2250298, NIH ES018274, ES015022.
A new biotelemetry system to monitor blood flow velocity, blood pressure and temperature in small animals: Preliminary data from cigarette smoke exposed SH rats
Authors: Maggie Lin , Griffiths Leigh G. , Uyeminami Dale , Johnson Katherine , Pinkerton Kent E. , Pitsillides Koullis
Published: June 2021

Telemetry monitoring of blood pressure and temperature in unrestrained rats is a relatively established procedure. However, monitoring blood pressure alone does not provide a comprehensive assessment of the physiologic changes occurring when animals are exposed to cigarette smoke. We have used a newly developed biotelemetry system capable of measuring, blood flow velocity, blood pressure and temperature in unrestrained SH rats.

The biotelemetry device (EG2‐V1S2TM2, Endosomatic Systems Inc, Davis, CA) was implanted in 340–380 g SH rats under inhalational general anesthesia. A 1.5mm diameter Doppler flowprobe was attached around the abdominal aorta just caudal to the renal artery and a 1F (330μm diameter) solid state pressure sensor was inserted in the abdominal aorta at the level of the aortic trifurcation. Preliminary data from this new system show that after 30 min of 60 mg/m3 cigarette smoke exposure, average systolic flow velocity increased by 33.5% and average systolic blood pressure increased by 47% of pre‐exposure levels.

The described biotelemetry system combines data from a Doppler flowprobe and a 1F high fidelity pressure sensor for making direct measurements of both blood flow velocity, blood pressure and temperature. This totally implantable telemetry system is suitable for small animals, such as rats, and provides new information regarding the effects of cigarette smoke on vascular resistance.
Effect of Acute Infusion of Low‐Molecular‐Weight Polyvinylpyrrolidone on Renal Function
Authors: Yan Qi , Du Zhaopeng , Wan Laxiang , Beck Franz-Xaver , Giebisch Gerhard , Wang Tong
Published: June 2021

Polyvinylpyrrolidone (PVP) is a linear polymer of 1‐vinyl‐2‐pyrrolidone monomers, used initially as a blood plasma expander and later in the pharmacy, food, beverage, and cosmetic industries. PVP is not readily degraded in the body and is sequestered in monocyte/macrophage systems, and in the kidney. Long‐term administration of PVP‐containing solutions causes PVP storage diseases and renal failure. In clearance experiments we examined the effect of acute i.v. infusion of PVP on renal function. Blood urea nitrogen (BUN), urinary beta‐N‐glucosaminidase (NAG), a lysosomal enzyme involved in the degradation of glycoprotein, and alpha 1‐microglobulin, an indicator of proximal tubule absorption ability, were also measured to determine whether acute PVP infusion causes renal injury. PVP infusion elicited significant diuretic and natriuretic effects and reduced glomerular filtration rate (GFR) by 37%. Urine volume, absolute (ENa) and fractional (FENa) Na+ excretion increased 5.5‐fold, 24‐fold and 51‐fold respectively. A relatively small kaliuretic effect was also observed (EK and FEK increased 0.55‐ and 1.8‐fold, respectively). After 2 h PVP infusion, BUN and NAG had not changed significantly (14.8 vs. 17.9 mg/dl and 0.11 vs. 0.18 mU/min/100g bw, respectively). Alpha 1‐microglobulin excretion increased 12‐fold (1.96 vs. 0.15 ng/min/100g bw), indicating that a large portion of fluid and Na+ loss is due to reduced absorption in the proximal tubule. The 24‐fold increase in ENa and the 12‐fold increase in alpha 1‐microglobulin excretion suggest that fluid and electrolyte absorption were also reduced in other nephron segments. We conclude that acute PVP infusion produces diuretic and natriuretic effects due to the osmotically induced reduction of proximal tubular absorption, but does not cause renal damage.
Quantitative phosphoproteomics of hypercalcemia induced nephrogenic diabetes insipidus
Authors: Khositseth Sookkasem , Somparn Poorichaya , Thippamon Nattakarn , Uawithya Panapat , Shen Rong-Fong , Chen Shu-Hui
Published: June 2021

Nephrogenic diabetes insipidus (NDI) could result from hypercalcemia (HC). The mechanism of NDI in parathyroid hormone induced HC was studied by using phosphoproteomic technique. Rats were infused with 30 μg/kg/day of PTH or vehicle for 1 day. Inner medulla collecting ducts from HC rats and control (C) were isolated. Tryptic phosphopeptides (immobilized metal affinity chromatography‐enriched) were identified and quantified by mass spectrometry (LTQ‐Orbitrap) using label‐free methodology. Phosphopeptide ratios were calibrated by total protein levels. Sixty nine and 180 phosphorylation sites were increased and decreased in HC, respectively. A down‐regulation of cytoskeletal proteins, nucleotide binding proteins, and transmembrane transporters were revealed. There was a significant decrease in aquaporin‐2(Aqp2) phosphorylation at S256 (by 60%) and S261 (by 50%), and urea transporter (Slc14a2) phosphorylation at S62 and S63 (by 80%) in HC. Immunoblots demonstrated 34% decrease of total Aqp2 (P=0.08), 60% decrease of pS256 (P<0.001), 181% increase of pS261 (P<0.05), and 30% decrease of total Slc14a2 (P<0.05) in the HC as compared to the C. In conclusion, two key proteins in urine concentrating mechanism, Aqp2 and Slc14a2, were found decreased in both phosphorylated forms in HC‐ induced NDI model. Other signaling molecules that might play important roles in this disease process have been identified.
A Mathematical Model for a Renal Medullary Tubule Structure of Neonatal Rats
Authors: Marcano Mariano , Bonilla-Félix Melvin
Published: June 2021

Our knowledge of the kidney in development, particularly its structure, is very limited. We used a mathematical model in conjunction with optimization problems to study the urine production capability of a medullary structure consisting of two populations of Henleˈs loops at different levels of development. In the model, the descending limb has heterogeneous transport properties along the medulla and the ascending limb only has a thick segment. The model parameters consist of membrane transport rates and boundary values for water flows and solute concentrations, and their baseline values were obtained from reported means in physiological experiments or they were estimated using physiological assumptions. By maximizing the solute free‐water absorption (FWA) and using parameter ranges from physiological experiments of neonatal rats in antidiuresis, we obtained results that yield a urine osmolality of 479 mOsmol/KgH2O and a urine flow of 0.126 nl/min per nephron. By minimizing the FWA and using parameter ranges from experiments of neonatal rats in diuresis, the results produced a urine osmolality of 75 mOsmol/KgH2O and a urine flow of 0.962 nl/min per nephron. These results are in agreement with the statement that the neonatal mammalian kidney can dilute urine as efficiently as the mature kidney, but its concentrating capability is limited.

Research supported by NIH‐NIGMS grant SC1GM084744.
Effects of low maternal aldosterone on offspring cardiovascular development
Authors: McKee Travis , Knoblich Penny
Published: June 2021

Maternal conditions are well known to affect fetal cardiovascular development. This study was designed to investigate in spontaneously hypertensive rats (SHR), the effects of maternal aldosterone suppression on the development of hypertension in the offspring. Right adrenalectomy was combined with cryodestruction of the zona glomerulosa of the left adrenal gland of SHR females. A sham procedure was performed on control rats. Rats were mated and offspring were studied at 10–13 weeks of age. Male rats were implanted with a PA‐C40 remote monitoring device placed into the femoral artery to record cardiovascular parameters for 4 weeks. Systolic (mmHg), diastolic (mmHg), and mean blood pressure (mmHg), heart rate (beats per minute), and activity levels were recorded. Data was sorted by activity level, and parameters were compared to evaluate same age data points between freeze and sham groups. No differences were found in any of the parameters between the offspring from sham and adrenal frozen maternal rats. These results suggest that lowered maternal plasma aldosterone levels do not adversely affect fetal cardiovascular development. This project was supported by funding from the Department of Biological Sciences, Minnesota State University, Mankato.
Angiotensin II receptor subtype 1a (AT1aR) gene silencing in neurons of the subfornical organ (SFO) prevents increased drinking behavior in bile duct ligated rats
Authors: Walch Joseph D. , Cunningham J. Thomas
Published: June 2021

Bile duct ligation (BDL) causes congestive liver failure that is associated with dilutional hyponatremia due to increased fluid intake and vasopressin release. In BDL rats, angiotensin II receptor subtype 1a (AT1aR) gene and protein expression is increased in the subfornical organ (SFO). This project tested the hypothesis that increased AT1aR expression in the SFO contributes to elevated drinking behavior in BDL rats. Rats were injected with an adeno‐associated viral vector containing GFP and either shRNA against AT1aR or scrambled RNA in the SFO. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were anesthetized 28 days after BDL surgery and perfused for immunohistochemical analysis and to verify the placement of the injections. BDL rats injected in the SFO with the control construct showed increased water intake compared sham ligated rats injected with either construct. In BDL rats injected in the SFO with AT1aR shRNA, this increase in water intake was significantly attenuated (RM Two‐way ANOVA followed by SNK post‐hoc, P<0.05, n=7–9 per group). These results indicate that angiotensin receptors in the SFO contribute to increased thirst in BDL rats.
TRPC4 expression in Supraoptic (SON) and Paraventricular (PVN) Magnocellular Neurosecretory Cells
Authors: Nedungadi Thekkethil Prashant , Walch Joseph D , Cunningham J. Thomas
Published: June 2021

The canonical transient receptor potential channel 4 (TRPC4) has been implicated in dendritic neurotransmitter release from thalamic neurons. We examined the expression of TRPC4 in magnocellular cells (MNCs) of the SON and PVN in normal rats and rats with bile duct ligation (BDL), a model of inappropriate vasopressin (AVP) release. Using immunohistochemistry (IHC) double labeling, we observed colocalization of TRPC4 with AVP in both the SON and PVN. In IHC studies using dendritic specific protein MAP2, TRPC4 was localized to the dendrites of MNCs. The numbers of TRPC4 positive cells in the SON was significantly increased after BDL (BDL 87 ± 8; SHAM 63 ± 5) and double IHC for TRPC4 and AVP showed increased colocalization in the SON but not in the PVN (BDL SON 56 ± 6; SHAM SON 36 ± 5; BDL PVN 31 ± 2; SHAM PVN 31 ± 5). Using quick immunohistochemistry (IHC) and laser capture microdissection (LCM) we captured 7–10 MNC AVP neurons for real‐time RT‐PCR measurement of TRPC4 mRNA in the PVN and the SON. TRPC4 transcriptional activity, as evaluated by 2−ΔΔCt, was significantly elevated in the SON of cirrhotic rats, but not in the PVN (BDL SON 5.33 ± 0.99; SHAM SON 1.3 ± 0.6; BDL PVN 0.6 ± 0.17; SHAM PVN 1.4 ± 0.5). These results confirm the expression of TRPC4 in AVP SON neurons and demonstrate that TRPC4 gene and protein expression is increased in the SON of BDL rats. R01 HL062579.
Na delivery, ENaC and mitochondrial NCX mediate flow‐stimulated collecting duct ET‐1 production
Authors: Pandit Meghana , Kohan Donald E
Published: June 2021

Collecting duct (CD) endothelin‐1 (ET‐1) inhibits Nareabsorption; its deficiency causes marked hypertension. CD ET‐1 is increased by volume expansion and fluid flow, suggesting coupling of CD Nadelivery to ET‐1 synthesis. To assess this, we studied the effect of Naon flow‐regulated ET‐1 production by mpkCCDc14 cells. Cells were subjected to static conditions or 2 dyne/cm2 for 2 hr, followed by determination of ET‐1 mRNA (N=7–12 all conditions). Flow with 300 mOsm/L NaCl increased ET‐1 mRNA levels to 165% of that seen under static conditions. Increasing media osmolarity to 450 mOsm/L with NaCl or sodium acetate, but not mannitol or choline Cl further augmented the ET‐1 flow response. Decreasing Na to 150 mOsm/L while adjusting to 300 mOsm/L with urea, mannitol or choline chloride reduced the flow response. Inhibition of ENaC with amiloride or benzamil prevented the ET‐1 flow response. Aldosterone almost doubled the flow response. Given that we have previously shown that calcium controls CD ET‐1 production, the effect of Na/Ca exchanger (NCX) blockade was assessed. SEA0400, a plasma membrane NCX inhibitor, did not affect the ET‐1 flow response. However, CGP37157, a mitochondrial NCX inhibitor, almost abolished the flow response. These data suggest that flow‐stimulated CD ET‐1 mRNA is mediated by Na entry via ENaC, resulting in mitochrondrial NCX‐induced increased [Ca]i and ultimately ET‐1 gene transcription.
Regulated intramembrane proteolysis contributes to control of the osmoprotective transcription factor, NFAT5
Authors: Izumi Yuichiro , Burg Maurice B. , Ferraris Joan D.
Published: June 2021

Nuclear Factor of Activated T‐Cells 5 (NFAT5, also TonEBP or OREBP) increases transcription of osmoprotective genes in response to hypertonicity. Regulated intramembrane proteolysis (RIP) is involved in a number of intracellular and extracellular signaling pathways. The present studies were aimed at testing for a possible role of RIP in controlling NFAT5 activity. RIP is activated by sheddases and intramembrane cleaving proteases (iCLiPs). We studied the effects of inhibitors of those proteases on NFAT5 transcriptional and transactivating activities, using luciferase reporters in HEK293 cells. We screened inhibitors of sheddases and iCLiPS, namely ADAM, MMP, β‐secretase, γ‐secretase, SPP, zinc metalloprotease, and rhomboid. Zinc metalloprotease inhibitor (1, 10 phenanthroline monohydrate) and rhomboid inhibitor (tosyl phenylalanine chloromethyl ketone: TPCK) inhibit hypertonicity induced NFAT5 transcriptional and transactivating activity in a dose dependent manner. Further, zinc metalloprotease inhibitor, but not rhomboid inhibitor, reduces hypertonicity‐induced increase of NFAT5 protein abundance. We conclude that RIP contributes to hypertonicity‐induced increase of NFAT5 activity. The research is funded by The Intramural Research Program of NHLBI, NIH.
Phospholipases A2 (PLA2) and cyclooxygenase 1 (COX‐1) are critical for angiotensin II (Ang‐II)‐induced reactive oxygen species (ROS) production and L‐type Ca2+ current in subfornical organ (SFO) neurons
Authors: Wang Gang , Anrather Josef , Pickel Virginia , Iadecola Costantino , Davisson Robin
Published: June 2021

We recently showed that cyclooxygenase 1 (COX‐1)‐derived prostaglandin E2 (PGE2) signaling through PGE2 type 1 receptors (EP1R) in the SFO is required for ROS‐mediated hypertension induced by slow‐pressor Ang‐II in vivo. However, the cellular signaling mechanisms remain unclear. Electron microscopy showed COX‐1 distribution in SFO dendrites and axon terminals. Ang‐II (100 nM) potentiated PGE2 release from freshly dissociated SFO cells (+ 209±55%, p<0.05 vs vehicle, n=4), an effect that was dependent on COX‐1. The general PLA2 inhibitor N‐(p‐amylcinnamoyl) anthranilic acid (ACA, 1 μM) inhibited Ang‐II (100 nM)‐induced superoxide (O2−•) production (+ 3±3%, p<0.05 vs vehicle, n=17) but not PGE2 (100 nM)‐induced O2−• production (+28.4±3.3%, p>0.05 vs vehicle, n=17) in SFO neurons. ACA (1 μM) also blocked Ang‐II‐induced potentiation of the voltage‐gated L‐type Ca2+ current (− 2.4±2%, p<0.05 vs vehicle, n=5), but not PGE2‐induced L‐type Ca2+ current (+42±4.2%, p>0.05 vs vehicle, n=5) in SFO neurons. This PGE2‐induced but ACA‐resistant L‐type Ca2+ current was completely inhibited by co‐application of the EP1R antagonist SC‐51089 (10 μM) (−7.7±5.2, p<0.05 vs vehicle, n=4). Our results suggest that Ang‐II increases O2−• and Ca2+ influx via PLA2‐ and COX‐1‐mediated production of PGE2 that acts on EP1R in SFO neurons.
Tonic and reflex control of renal sympathetic nerve activity are altered by a discrete increase in sodium intake in renovascular hypertensive rats
Authors: Shimoura Caroline Gusson , Lincevicius Gisele Silvério , Bergamaschi Cassia , Campos Ruy
Published: June 2021

Sympathoexcitation and baroreceptor dysfunction are involved in the pathophysiology of renovascular hypertension (2K1C). In the present study we investigated the effects of a discrete increase in sodium intake in 2K1C rats. Animals were divided in 4 independent groups (n=± 6/group) and treated for 2 weeks with normal sodium diet (0.5% NNa+) or high sodium diet (2% HNa+) after 4 weeks of renovascular hypertension. Water intake (WI) and urinary volume (UV) was increased in 2K1C HNa+ compared to 2K1C NNa+ (WI 2K1C HNa+ 61.1±6.4 2K1C; NNa+39.8±2.5; mL/day); UV (2K1C HNa+ 50 ± 5 mL/day; NNa+ 25.4±2.3; 2K1C). Arterial blood pressure (ABP) was significantly increased in 2K1C compared only to control group (2K1C NNa+ 174 ± 5; control NNa+ 100 ± 3 mmHg). Increased sodium intake enhanced the baroreflex sensitivity gain of HR in control and 2K1C (control NNa+ −2.6±0.7; control HNa+−5.3±0.4; 2K‐1C NNa+ −1.7±0.2; 2K‐1C HNa+ −4.1±0.6 bpm/mmHg). Renal sympathetic nerve activity (RSNA) gain was enhanced only in 2K1C HNa+(2K1C NNa+ −0.7±0.03; 2K1C HNa+ −1.4±0.06 pps/mmHg). Our results suggest that in the renovascular hypertension the improvement in the baroreceptor sensitivity to the kidney is probably a mechanism leading to increased diuresis avoiding further increase in ABP.

Supported by FAPESP, CNPq
Pressure–natriuretic and hemodynamic adjustments to chronic ACE inhibition in inbred F344 and Lewis rat strains
Authors: Menzies Robert Ian , Kenyon Christopher J , Mullins John J , Bailey Matthew A
Published: June 2021

Genetic background has a major influence on the severity of renovascular disease. Previously we showed that Lewis rats with inducible hypertension caused by transgenic renin expression have lower blood pressure, higher renal plasma flow (RPF) and glomerular filtration rate (GFR) and are relatively protected from hypertensive renal microvascular injury compared to Fisher (F344) rats. Using a congenic/consomic approach we determined that a polymorphism of the angiotensin converting enzyme (ACE) gene is a major contributory factor in explaining these strain differences.

In the current study we have compared renal function in anaesthetised, non transgenic F344 and Lewis rats. Pressure‐natriuresis (PN) and pressure‐renal blood flow (PRBF) relationships were measured in groups of rats pre‐treated with and without Captopril (80mg/L drinking water 1–2wks) to inhibit ACE activity. Control F344 rats had higher baseline blood pressure and poorer PN compared with Lewis (ie shallower slope of PN curve). Captopril treatment significantly improved RBF without affecting PN in F344 rats. Conversely, Captopril reduced the slope of the PN curve in Lewis rats without affecting RBF responses to pressure. Taken together these results indicate a dominant vascular response to ACE inhibition in F344 compared with a dominant tubular response in Lewis rats.