Female Spontaneously Hypertensive Rats Research Articles

Sustained activation of 12/15 lipoxygenase (12/15 LOX) contributes to impaired renal recovery post ischemic injury in male SHR compared to females

BackgroundAcute kidney injury (AKI) due to ischemia-reperfusion (IR) is a serious and frequent complication in clinical settings, and mortality rates remain high. There are well established sex differences in renal IR, with males exhibiting greater injury following an ischemic insult compared to females. We recently reported that males have impaired renal recovery from ischemic injury vs. females. However, the mechanisms mediating sex differences in renal recovery from IR injury remain poorly understood. Elevated 12/15 lipoxygenase (LOX) activity has been reported to contribute to the progression of numerous kidney diseases. The goal of the current study was to test the hypothesis that enhanced activation of 12/15 LOX contributes to impaired recovery post-IR in males vs. females.Methods13-week-old male and female spontaneously hypertensive rats (SHR) were randomized to sham or 30-minute warm bilateral IR surgery. Additional male and female SHR were randomized to treatment with vehicle or the specific 12/15 LOX inhibitor ML355 1 h prior to sham/IR surgery, and every other day following up to 7-days post-IR. Blood was collected from all rats 1-and 7-days post-IR. Kidneys were harvested 7-days post-IR and processed for biochemical, histological, and Western blot analysis. 12/15 LOX metabolites 12 and 15 HETE were measured in kidney samples by liquid chromatography–mass spectrometry (LC/MS).ResultsMale SHR exhibited delayed recovery of renal function post-IR vs. male sham and female IR rats. Delayed recovery in males was associated with activation of renal 12/15 LOX, increased renal 12-HETE, enhanced endoplasmic reticulum (ER) stress, lipid peroxidation, renal cell death and inflammation compared to females 7-days post-IR. Treatment of male SHR with ML355 lowered levels of 12-HETE and resulted in reduced renal lipid peroxidation, ER stress, tubular cell death and inflammation 7-days post-IR with enhanced recovery of renal function compared to vehicle-treated IR male rats. ML355 treatment did not alter IR-induced increases in plasma creatinine in females, however, tubular injury and cell death were attenuated in ML355 treated females compared to vehicle-treated rats 7 days post-IR.ConclusionOur data demonstrate that sustained activation 12/15 LOX contributes to impaired renal recovery post ischemic injury in male and female SHR, although males are more susceptible on this mechanism than females.

Memory deficits and hippocampal cytokine expression in a rat model of ADHD

Attention-deficit/hyperactivity disorder (ADHD) is a complex behavioral disorder characterized by hyperactivity, impulsivity, inattention, and deficits in working memory and time perception. While animal models have advanced our neurobiological understanding of this condition, there are limited and inconsistent data on working and elapsed time memory function. Inflammatory signaling has been identified as a key factor in memory and cognitive impairments, but its role in ADHD remains unclear. Additionally, the disproportionate investigation of male subjects in ADHD research has contributed to a poor understanding of the disorder in females. This study sought to investigate the potential connections between memory, neuroimmunology, and ADHD in both male and female animals. Specifically, we utilized the spontaneously hypertensive rat (SHR), one of the most extensively studied animal models of ADHD. Compared to their control, the Wistar-Kyoto (WKY) rat, male SHR are reported to exhibit several behavioral phenotypes associated with ADHD, including hyperactivity, impulsivity, and poor sustained attention, along with impairments in learning and memory. As the hippocampus is a key brain region for learning and memory, we examined the behavior of male and female SHR and WKY rats in two hippocampal-dependent memory tasks. Our findings revealed that SHR have delay-dependent working memory deficits that were similar to, albeit less severe than, those seen in hippocampal-lesioned rats. We also observed impairments in elapsed time processing in female SHR, particularly in the discrimination of longer time durations. To investigate the impact of inflammatory signaling on memory in these rats, we analyzed the levels of several cytokines in the dorsal and ventral hippocampus of SHR and WKY. Although we found some sex and genotype differences, concentrations were generally similar between groups. Taken together, our results indicate that SHR exhibit deficits in spatial working memory and memory for elapsed time, as well as some differences in hippocampal cytokine concentrations. These findings contribute to a better understanding of the neurobiological basis of ADHD in both sexes and may inform future research aimed at developing effective treatments for the disorder. Nonetheless, the potential mediating role of neuroinflammation in the memory symptomatology of SHR requires further investigation.

Sex-Specific Responses to Tacrolimus and Mycophenolate Mofetil in Spontaneously Hypertensive Rats

In recent decades, the roles of tacrolimus and mycophenolate mofetil (MMF) in hypertension have been under discussion. However, the question of whether there are sex-specific responses to these agents has not received enough attention. Aim: To evaluate sex-specific differences in the responses to tacrolimus and mycophenolate mofetil in female (F) and male (M) spontaneously hypertensive rats (SHRs) and evaluate whether T cells contribute to mean arterial pressure (MAP) changes. Methods: Male and female SHRs received either tacrolimus or MMF for 14 days. The rats were implanted with radiotelemeters. MAP was measured chronically; then, circulating and renal infiltrated CD4+, CD8+, T helper 17 (Th17), and T regulatory (Treg) cells were quantified using flow cytometry. Key Findings: Tacrolimus increased MAP only in males, and it decreased CD4+ and CD8+ T cells in both males and females (p < 0.05). The tacrolimus-induced reduction of renal CD4+ and Treg cells was more profound in males. MMF reduced MAP and circulating and renal CD4+ and CD8+ T cells in the male and female rats. MMF also decreased Th17 and Treg cells in both sexes, but the decrease in Th17 was higher in males (p < 0.05) and the reduction in Treg cells was higher in females (p < 0.05). Our findings indicate that the effects of tacrolimus and MMF on renal T cell subsets are sex-specific. Significance: Targeting T cells in hypertension using therapeutic agents may have different effects on men and women; so, the management of hypertension and post-transplant hypertension using these agents should be specified by gender.

Effects of adolescent methylphenidate administration on methamphetamine conditioned place preference in an animal model of attention-deficit/hyperactivity disorder: Examination of potential sex differences

BackgroundIndividuals with attention-deficit/hyperactivity disorder (ADHD) are more likely to be diagnosed with a substance use disorder; however, the effects of long-term psychostimulant treatment on addiction are mixed. Preclinical studies are useful for further elucidating the relationship between ADHD and addiction-like behaviors, but these studies have focused on male subjects only. The goal of the current study was to determine if early-life administration of methylphenidate (MPH) augments methamphetamine (METH) conditioned place preference (CPP) and/or potentiates reinstatement of CPP in both male and female rats. MethodsMale and female spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) received either MPH (1.5mg/kg; p.o.) or vehicle (1.0ml/kg) during adolescence (postnatal day [PND] ~29–57). Two weeks after cessation of MPH treatment, rats were tested for METH CPP (1.0mg/kg or 2.0mg/kg; s.c.). Rats were then given extinction sessions. Once rats met extinction criteria, they were tested for reinstatement of CPP following a priming injection of METH (0.25mg/kg; s.c.). ResultsAll groups developed METH CPP, except vehicle-treated SHR males and vehicle-treated WKY females conditioned with the higher dose of METH (2.0mg/kg). Female SHRs treated with MPH showed greater reinstatement of METH CPP compared to female SHRs treated with vehicle. Adolescent MPH treatment did not augment the locomotor-stimulant effects of METH in adulthood. ConclusionsThese results demonstrate the importance of considering biological sex when prescribing psychostimulant medications for ADHD as long-term MPH administration may increase the risk of continued drug use in females with ADHD following a period of abstinence.

Abstract 096: Effects Of Chronic Hypertension And Sex On Spatial Reference And Working Memory

Chronic hypertension is a primary risk factor of vascular dementia. The hippocampus is a brain region critical to learning and memory and is highly susceptible to hypoxia/ischemia. Here, we investigated how chronic hypertension affects two different types of memory function, and whether one sex is more susceptible to hypertension-induced memory impairment. Memory function in adult male and female SHR (spontaneously hypertensive rats) and normotensive Wistar rats was tested using two behavioral tasks (n=6/group). The radial arm maze (RAM) tested reference memory that is largely hippocampal-dependent by measuring the time it took to retrieve rewards from three baited arms during daily 10 min sessions. A continuous y maze task was used to calculate alternation index and determine working memory that is cortico-hippocampal-dependent. Data are mean±SEM and comparisons made via a two-way ANOVA with a post-hoc Tukey test. In the RAM task, female rats had steeper learning curves than males as they completed the task in less time with each session (Fig 1). There was a significant interaction between sex and hypertension in the y maze task (F (1,20) =13.26, p=0.002). Working memory was impaired in female SHR, with a lower alternation index (index=0.60±0.03) than Wistar rats (index=0.74±0.03). However, male SHR that had a higher alternation index of 0.75±0.02 compared to 0.58±0.6 in male Wistar rats. Overall, females had stronger reference memory than males regardless of hypertension. Working memory was differentially affected in male and female SHR, suggesting sex differences in the susceptibility of different brain regions and types of memory to hypertension-induced dysfunction.

Abstract P3103: Heart Failure In Menopause: Treatment With Rock Inhibitor And Atorvastatin

In advanced age, women tend to develop heart failure with preserved ejection fraction (HFpEF) due to estrogen depletion. The signaling pathway of ROCK is involved in cardiac complications consequent to female aging. Thus, this work investigated the comparative effects of atorvastatin and ROCK inhibitor, fasudil on the treatment of HFpEF induced by oophorectomy (OVX) in spontaneously hypertensive rats (SHR). Protocols were approved by the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Under anesthesia with ketamine (80 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.), female SHR were submitted to OVX procedure and after 8 wks were randomly orally treated with either vehicle or atorvastatin (40 mg/kg) or fasudil (10 mg/kg) during 4 wks. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and LV catheterization. Cardiac tissues were collected for histologic analysis. Cardiac hypertrophy was reversed by treatment with atorvastatin or fasudil. Increased systolic pressure of LV was observed in SHR-OVX group (149.2 ± 7.9 mmHg), which was partially reduced by fasudil (123.4 ± 7.4 mmHg) and normalized by atorvastatin (94.5 ± 3.9 mmHg). Reduction of the increased diastolic dysfunction observed by echocardiogram was confirmed, since the recovery of the increased LV end diastolic pressure of SHR-OVX (32.1 ± 1.8 mmHg) occurred after treatment with atorvastatin (22.4 ± 9.6 mmHg) and fasudil (11.9 ± 2.4 mmHg). Fibrosis was observed because of the increased collagen deposition in SHR-OVX (6.2 ± 0.1%), with partial reduction with fasudil (4.4 ± 0.3%) and normalization after treatment with atorvastatin (1.9 ± 0.7%). Cell proliferation was evaluated using the p38 marker, where the percentage of nuclei marked was determined in relation to the total, through immunohistochemical technique. An increase in nuclear p38 in the SHR-OVX group was observed (23.2 ± 3.2%), however, the treatment with atorvastatin (12.1 ± 1.7%) and fasudil (5.3 ± 1.3%) reduced the nuclear p38 detection. In conclusion, atorvastatin and fasudil promoted recovery of HFpEF induced by estrogen depletion in SHR. Inhibition of ROCK signalling by atorvastatin could be involved in the reduction of diastolic dysfunction observed in OVX-SHR group.

Oat Beta-Glucan Alone and in Combination with Hydrochlorothiazide Lowers High Blood Pressure in Male but Not Female Spontaneously Hypertensive Rats.

Oats are considered a functional food due to the beneficial health effects associated with their consumption and are suitable to be explored for their ability to prevent or manage chronic disease, such as hypertension. Here, we examined the cardiovascular benefits of an oat beta-glucan extract in male and female spontaneously hypertensive rats (SHRs) to unravel its sex-specific roles when used with an anti-hypertensive medication, hydrochlorothiazide. Five-week-old male and female SHRs and Wistar-Kyoto (WKY) rats were treated with oat beta-glucan and hydrochlorothiazide for 15 weeks. Twenty-week-old male and female SHRs showed high blood pressure (BP), cardiac remodeling, and cardiac dysfunction. These animals also had significantly increased levels of malondialdehyde (MDA), angiotensin II, and norepinephrine. Treatments with beta-glucan and hydrochlorothiazide were able to differentially prevent high BP, cardiac dysfunction, and alterations in malondialdehyde (MDA), angiotensin II, and norepinephrine in 20-week-old male and female SHRs. To conclude, beta-glucan alone and in combination with hydrochlorothiazide may be a promising a strategy for managing hypertension and related cardiac complications.

Functional symmetry of the aortic baroreflex in female spontaneously hypertensive rats.

Altered baroreflex function is well documented in hypertension; however, the female sex remains far less studied compared with males. We have previously demonstrated a left-sided dominance in the expression of aortic baroreflex function in male spontaneously hypertensive rats (SHRs) and normotensive rats of either sex. If lateralization in aortic baroreflex function extends to hypertensive female rats remains undetermined. This study, therefore, assessed the contribution of left and right aortic baroreceptor afferents to baroreflex modulation in female SHRs. Anesthetized female SHRs (total n = 9) were prepared for left, right and bilateral aortic depressor nerve (ADN) stimulation (1-40 Hz, 0.2 ms, 0.4 mA for 20 s) and measurement of reflex mean arterial pressure (MAP), heart rate (HR), mesenteric vascular resistance (MVR) and femoral vascular resistance (FVR). All rats were also matched for the diestrus phase of the estrus cycle. Reflex (%) reductions in MAP, HR, MVR and FVR were comparable for both left-sided and right-sided stimulation. Bilateral stimulation evoked slightly larger ( P = 0.03) reductions in MVR compared with right-sided stimulation; however, all other reflex hemodynamic measures were similar to both left-sided and right-sided stimulation. These data show that female SHRs, unlike male SHRs, express similar central integration of left versus right aortic baroreceptor afferent input and thus show no laterization in the aortic baroreflex during hypertension. Marginal increases in mesenteric vasodilation following bilateral activation of the aortic baroreceptor afferents drive no superior depressor responses beyond that of the unilateral stimulation. Clinically, unilateral targeting of the left or right aortic baroreceptor afferents may provide adequate reductions in blood pressure in female hypertensive patients.

Noninvasive Monitoring of Blood Pressure and Heart Rate during Estrous Cycle Phases in Normotensive Wistar-Kyoto and Spontaneously Hypertensive Female Rats.

Since 2015, the National Institutes of Health has called for its funded preclinical research to include both male and female subjects. However, much of the basic animal research that has studied heart rate and blood pressure in the past has used male rats. Male rats have been preferred for these studies to avoid the possible complicating effects of the female estrous cycle. The aim of the current study was to determine whether blood pressure and heart rates vary as a function of the estrous cycle phase of young normotensive Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) female rats. Blood pressure and heart rate were measured at the same time of day throughout the estrous cycle by using a noninvasive tail cuff sphygmomanometric technique. As expected, 16-wk-old female SHR rats had higher blood pressure and heart rates than did age-matched female WKY rats. However, no significant differences in mean, systolic, or diastolic arterial blood pressure or heart rate were detected across the different stages of the estrous cycle in either strain of female rats. Consistent with previous reports, heart rates were higher and showed less variation in the hypertensive SHR female rats as compared with the normotensive WKY female rats. These results indicate that studies measuring blood pressure and heart rate can include young female SHR and WKY rats with no effect of estrous cycle stage.

Sustained mitochondrial dysfunction in male, but not female Spontaneous Hypertensive Rats (SHR) corresponds with delayed renal recovery from ischemic injury

Acute kidney injury (AKI) due to ischemia-reperfusion (IR) is a serious and frequent complication in hospital settings with high mortality rates. The mechanisms mediating renal IR injury and leading to increased risk of later developing cardiovascular and renal diseases in either sex remain poorly understood. We previously reported that there is sex difference in renal recovery from IR where males have a delayed recovery vs females. Mitochondrial dysfunction plays a pivotal role in the pathogenesis of AKI in males, resulting in abnormal kidney repair and disease progression. However, the impact of IR on mitochondrial function in females has not been examined. The goal of the current study was to test the hypothesis that IR results in greater sustained mitochondrial dysfunction in males vs. females corresponds to delayed recovery of renal function in males. 13-week-old male and female SHR were subjected to sham or 30-minute warm bilateral IR. Subsets of rats were euthanized 1- or 7-days post-IR (n=6/group). Blood was collected to measure kidney function. The right kidney was dissected and processed for electron microscopy (EM) to assess mitochondrial structure, histological analysis of renal structure, and measurement of mitochondrial DNA (mtDNA) copy number via RT-PCR. Tubular cells were isolated from the left kidney to measure oxygen consumption rate (OCR) to assess mitochondrial respiration using Seahorse XFe24 Extracellular Flex Analyzer. IR increased plasma creatinine (Pcr) in male and female SHR vs. respective sham controls 1-day post-IR (PIR =0.0001; Psex✕IR=0.2). Pcr remained elevated in male, but not female, SHR 7 days post-IR (Pcr: PIR =0.03; Psex✕IR=0.04). OCR was reduced in male and female tubules 1 day post IR (PIR =0.001; Psex=0.94) and remained reduced only in tubules from males 7 days post-IR (PIR =0.0001; Psex✕IR=0.0001). Consistent with decreased OCR, EM revealed swollen round mitochondria, reduced mitochondria density with complete loss of cristae membranes vs. sham controls 1-day post-IR in both sexes, which was sustained 7 days post-IR only in males. Mitochondrial DNA copy number and peroxisome proliferator-activator receptor-y coactivator (PGC-1α) mRNA expression was also reduced 1-day post-IR in both sexes (PIR =0.05; Psex✕IR=0.59) and remained down only in males 7 days post-IR (PIR =0.02; Psex✕IR=0.001). In conclusion, our data demonstrate that there is sex difference in mitochondria after renal IR. Males have sustained mitochondrial dysfunction with delayed renal recovery compared to females. Further studies will be carried out to better understand the direct impact of mitochondria dysfunction on renal recovery post-IR in male and females. This work was supported by career development grant from AHA (20CDA35310362) to R Mohamed; National Institutes of Health (1P01HL134604-01 and R01 HL127091 to J. C. Sullivan. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypertension Reduces Aortic Strain in Both Sexes, but only Aorta from Female Rats Showed Increase in Stress: The Role of Appropriate Normalization Method to Evaluate Strain-Stress Parameters

The gold standard of arterial stiffness measurements in isolated aorta is the stress-strain (SS) relationship. Wall thickness (WT) and artery inner diameter (ID) are variables that plot the SS curve and should be normalized to the dimensions of the individual subject. Previously, we used measurements from the current literature, but the data collected were not accurate. Therefore, we hypothesized that the measurements of the vascular structure using echocardiogram (ECHO) should be applied to evaluate tissue puller to obtain SS data. With that, we also hypothesized that aorta from spontaneously hypertensive rats (SHR) has greater stress and lower strain levels than normotensive Wistar rats. To test this hypothesis, thoracic aorta parameters from 3-month-old male Wistar (WM) (body weight: 449±6.87g, n=4), SHR male (SM) (body weight: 295±3.34g, n=4), Wistar Female (WF) (body weight: 250±8.50g, n=4), and SHR Female (SF 169.4±2.69g, n=4) rats were measured from an ECHO. After, animals were killed and aortas were cut in 2 mm segments and mounted on Tissue Puller to measure SS. The inner diameter (ID) and wall thickness (WT) were measured by the ECHO for all animals: [WM (WT: 0.519±0.014mm, ID: 2.042±0.11mm), SM (WT: 0.485±0.026mm, ID: 2.023±0.055mm), WF (WT: 0.504±0.01mm, ID: 1.628±0.092mm), SF (WT: 0.521±0.011mm, ID: 1.64±0.099mm)] and there were no significant difference between these values. We then normalized the SS curves to each of the arteries’ parameters for ECHO and we found there were significant decreases in the strain values in aorta from hypertensive rats, regardless of sex (WM: 1.775±0.108 vs SM: 1.370±0.058; p=0.0146), and (WF: 2.056±0.218 vs. SF: 1.713±0.224; p=0.0517). We found a trend in the stress from female rats (WF: 1367±61 mN/mm2 vs SF 2361±391 mN/mm2; p=0.0597). There were no differences between male rats (WM:1.269±153 mN/mm2 vs SM: 1319±80 mN/mm2; p=0.671).Overall, these data show that ECHO should be used to accurately measure rat aorta structural parameters under the tissue puller. They also show SHR and Wistar were different in both sexes in strain, but only female SHR vs Wistar showed differences in stress. National Institutes of Health - R00GM118885, R01HL149762, and R00HL151889. and McNair Junior Fellows (MJF) Program This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

COVID-19: Understanding the impact of anti-hypertensive drugs and hydroxychloroquine on the ACE1 and ACE2 in lung and adipose tissue in SHR and WKY rats.

Hypertensive individuals taking anti-hypertensive drugs from renin-angiotensin system inhibitors may exhibit a more severe evolution of the disease when contracting the SARS-CoV-2 virus (COVID-19 disease) due to potential increases in ACE2 expression. The study investigated ACE1 and ACE2 axes and hydroxychloroquine in the lungs and adipose tissue of male and female normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). SHRs were treated with losartan (10mg/kg/day) or captopril (10mg/kg/day) for 14 days or 7 days with hydroxychloroquine (200 mg/kg/day) in drinking water. WKY rats were also treated for 7 days with hydroxychloroquine. Blood pressure (BP), protein, and mRNA expression of ACE1 and ACE2 were analyzed in serum, adipose, and lung tissues. Losartan and captopril reduced BP in both sexes in SHR, whereas hydroxychloroquine increased BP in WKY rats. Losartan reduced ACE2 in serum and lungs in both sexes and in adipose tissue of male SHRs. Captopril decreased ACE2 protein in the lung of females and in adipose tissue in both sexes of SHRs. Hydroxychloroquine decreased ACE1 and ACE2 proteins in the lungs in both sexes and adipose tissue in male SHRs. In female WKY rats, ACE2 protein was lower only in the lungs and adipose tissue. Losartan effectively inhibited ACE2 in male and captopril in female SHRs. Hydroxychloroquine inhibited ACE2 in male SHRs and female WKY rats. These results further our understanding of the ACE2 mechanism in patients under renin-angiotensin anti-hypertensive therapy and in many trials using hydroxychloroquine for COVID-19 treatment and potential sex differences in response to drug treatment.

Egg white protein hydrolysate decreased blood pressure via the competing endogenous RNA regulatory networks in female spontaneously hypertensive rats.

Despite numerous studies having reported the effects and mechanisms of antihypertensive peptides including peptides derived from egg white proteins, the role of peptides in a female hypertensive animal model is unknown. On the other hand, the role of epigenetic modulation by peptide treatment has rarely been investigated. This study sought to investigate the effect of egg white protein hydrolysate (EWH) in female spontaneously hypertensive rats (SHRs) as well as to explore the underlying mechanisms from the perspectives of the transcriptome and the profiles of non-coding RNAs. Young (12-14-week-old) female SHRs were orally administered 250 mg per kg body weight (low-dose) or 1000 mg per kg body weight (high-dose) EWH daily for 10 weeks. The blood pressure of the rats was monitored weekly. The mRNA and non-coding RNAs (miRNA, lncRNA, and circRNA) in the aorta were profiled by the high-throughput RNA-seq technique. Differentially expressed (DE) RNAs in the aorta were identified for the construction of the competing endogenous RNA (ceRNA) networks and key molecules were validated by qRT-PCR. The treatment of the high-dose EWH showed a significant effect on reducing blood pressure in female SHRs. Bioinformatic analyses revealed 813, 90, 347 and 869 DE-mRNAs, DE-miRNAs, DE-lncRNAs and DE-circRNAs, respectively. The CNTN5-LncRNA-XR_001835895.1-miR-384-5p was identified as the central network which was validated in the aorta and circulation of female SHRs. The results from this study demonstrated that the treatment with EWH reduced blood pressure via regulating the ceRNA networks in female SHRs, which provided novel insights into the mechanisms of food protein-derived antihypertensive peptides.

Diuretic and Natriuretic Effects of Hesperidin, a Flavanone Glycoside, in Female and Male Hypertensive Rats

Hesperidin (HSP) is a major flavanone glycoside in citrus fruits, including sweet oranges and lemons. It demonstrates numerous pharmacological activities, such as antihypertensive effects and cardiac and kidney tissue protection. However, its effect on modulating renal function has yet to be properly explored. Female and male Wistar spontaneously hypertensive rats (SHR) were used to test the effect of HSP on renal function. The rats were divided into different groups, treated orally, and placed in metabolic cages for urine collection for 8 h. HSP, at doses of 0.3-3 mg/kg, led to an increase in urine volume in both female and male SHR. This effect was associated with increased Na+ elimination (3 mg/kg) without causing any change in K+ excretion or pH and conductivity values. When given HSP in combination with hydrochlorothiazide (HCTZ) or amiloride (AMLR), urine volume and Na+ elimination were significantly increased compared to the group that received only HSP. In relation to K+ excretion, the depleting effect of HCTZ and the sparing of AMLR prevailed in both groups. Pre-treatment with a non-selective cholinergic receptor antagonist, atropine, partially prevented HSP-induced diuresis and natriuresis in male SHR, but this effect was not demonstrated with the non-selective inhibitor of the enzyme cyclooxygenase, indomethacin. This study shows the diuretic action of HSP in hypertensive rats, an activity probably associated with the cholinergic pathway. Although various biological actions have already been defined for HSP, this pioneering research reveals its potential as a diuretic medicine.

The role of transforming growth factor- in hypertension-induced cerebrovascular remodeling

Background: Hypertension (HT) promotes structural and functional changes in the cerebral microcirculation that can provoke irreversible cerebrovascular injury, leading to neuronal loss and brain atrophy,1 cognitive impairment, vascular dementia,2 and Alzheimer’s disease.3 Currently, the mechanisms and consequences of such remodeling are not fully understood. Transforming growth factor (TGF) is a morphogen that regulates cellular differentiation, induces endothelial-to-mesenchymal transition (EndoMT), and works as a contractile-to-synthetic switch in vascular smooth muscle cells (VSMC) phenotype.4 Plasma levels of TGF are increased in HT patients, and in spontaneously hypertensive rats (SHR) that exhibit vascular fibrosis.5 Although coincidental, causal roles through which elevated TGF may drive cerebrovascular remodeling in the HT brain are suspected, but unproven. We hypothesize that HT-induced TGF drives cerebrovascular remodeling, which decreases blood-brain barrier and impairs cerebrovascular autoregulation.
 Methods: Brain cortices and penetrating cerebral microvessels (BMVs) were isolated from male and female SHR and Wistar-Kyoto rats (WKY, control), and subjected to western blotting and immunofluorescence labeling for endothelial cell (EC) and VSMC differentiation markers, TGF canonical pathway markers SMAD2, 3 and 4, as well as basement membrane protein expression, and glial fibrillary acidic protein (GFAP) as a marker of astrocyte inflammation. Additionally, TGF-treated rat retinal microvascular endothelial cells (RRMECs), and human brain microvascular endothelial cells (hCMEC/D3) ± the TGF inhibitor vactosertib, were also subjected to western blotting and immunofluorescence labeling to assess endothelial cell (EC) and VSMC differentiation markers, as well as basement membrane protein expression.
 Results: TGF was significantly increased in SHR BMVs. Moreover, GFAP levels were significantly increased in the SHR cortex. The EC junctional proteins platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin), were significantly decreased in SHR. In contrast, CRBP-1, a marker for synthetic VSMC, and collagen IV and fibronectin levels significantly increased in SHR. Interestingly, female SHR rats had significantly increased levels of SMAD2/3, which was not evident in male SHRs, indicating a possible role for the non-canonical TGF pathway in male SHRs. Additionally, TGF significantly increased -smooth muscle actin (-SMA) and decreased VE-cadherin expression in RRMECs and D3 cells, consistent with EndoMT. TGF inhibition with vactosertib reversed these effects and significantly suppressed -SMA, and maintained VE-Cadherin similar to control RRMECs and D3s.
 Conclusions: We now have strong evidence for HT-induced cerebrovascular remodeling, where TGF mediates loss of both smooth muscle and endothelial differentiated phenotypes. The reversal of these effects using TGF blockers suggests that therapy to modulate TGF may represent a means to control HT-induced cerebrovascular remodeling.

Sex differences in apoptosis do not contribute to sex differences in blood pressure or renal T cells in spontaneously hypertensive rats.

Apoptosis is a physiological and anti-inflammatory form of cell death that is indispensable for normal physiology and homeostasis. Several studies have reported aberrant activation of apoptosis in various tissues at the onset of hypertension. However, the functional significance of apoptosis during essential hypertension remains largely undefined. The current study was designed to test the hypothesis that apoptosis contributes to sex differences in blood pressure and the T cell profile in spontaneously hypertensive rats (SHR). Apoptosis was measured in kidney, aorta and spleen of 13-week-old adult hypertensive male and female SHR. Female SHR had greater renal and aortic apoptosis compared to age-matched males; apoptosis in the spleen was comparable between the sexes. Based on well-established sex differences in hypertension, we tested the hypothesis that greater apoptosis in female SHR contributes to the lower BP and pro-inflammatory profile compared to males. Male and female SHR were randomized to receive vehicle or ZVAD-FMK, a cell permeable pan-caspase inhibitor, in established hypertension from 13 to 15 weeks of age or at the onset of hypertension from 6 to 12 weeks or age. Treatment with ZVAD-FMK lowered renal apoptosis in both studies, yet neither BP nor renal T cells were altered in either male or female SHR. These results suggest that apoptosis does not contribute to the control or maintenance of BP in male or female SHR or sex differences in renal T cells.

The influence of chromosome 4 on high ethanol consumption and blood pressure

The Spontaneously Hypertensive Rats (SHR) strain was developed through selective breeding for high systolic blood pressure. In our laboratory, we established a congenic rat strain named SHR.Lewis-Anxrr16 (SLA16). The SLA16 rat strain is genetically identical to the SHR except for the inserted Anxrr16 region in chromosome 4. Our objective was to evaluate the influence of this genomic region on ethanol consumption and blood pressure. First, we exposed SHR and SLA16 male and female rats to ethanol consumption. Results showed that, regardless of strain, females consumed more ethanol than males during forced (10% v/v) and spontaneous ethanol consumption (SEC; 2.5-20% v/v). Then, females from both strains were used to evaluate sensitivity to ethanol. No strain differences in the loss of righting reflex were observed after ethanol treatment (3g/kg, 20% w/v, intraperitoneal [i.p.]). But, in the triple test, female SHR rats presented lower sensitivity to the ethanol (1.2g/kg, 14% w/v, i.p.). Surprisingly, female SHR rats also presented higher blood pressure after SEC (10% v/v). Finally, losartan treatment was effective in decreasing the blood pressure of female rats of both strains, but had specific effects on SHR ethanol consumption. Our data suggest that SLA16 female rats consume less ethanol (10%), are more sensitive to its effects, and present lower blood pressure than SHR female rats. We demonstrated that the Anxrr16 locus in chromosome 4 is a genetic candidate to explain high ethanol consumption and blood pressure, at least in females.

Cardiac Cx43 Signaling Is Enhanced and TGF-β1/SMAD2/3 Suppressed in Response to Cold Acclimation and Modulated by Thyroid Status in Hairless SHRM.

The hearts of spontaneously hypertensive rats (SHR) are prone to malignant arrhythmias, mainly due to disorders of electrical coupling protein Cx43 and the extracellular matrix. Cold acclimation may induce cardio-protection, but the underlying mechanisms remain to be elucidated. We aimed to explore whether the adaptation of 9-month-old hairless SHRM to cold impacts the fundamental cardiac pro-arrhythmia factors, as well as the response to the thyroid status. There were no significant differences in the registered biometric, redox and blood lipids parameters between hairless (SHRM) and wild type SHR. Prominent findings revealed that myocardial Cx43 and its variant phosphorylated at serine 368 were increased, while an abnormal cardiomyocyte Cx43 distribution was attenuated in hairless SHRM vs. wild type SHR males and females. Moreover, the level of β-catenin, ensuring mechanoelectrical coupling, was increased as well, while extracellular matrix collagen-1 and hydroxyproline were lower and the TGF-β1 and SMAD2/3 pathway was suppressed in hairless SHRM males compared to the wild type strain. Of interest, the extracellular matrix remodeling was less pronounced in females of both hypertensive strains. There were no apparent differences in response to the hypothyroid or hyperthyroid status between SHR strains concerning the examined markers. Our findings imply that hairless SHRM benefit from cold acclimation due to the attenuation of the hypertension-induced adverse downregulation of Cx43 and upregulation of extracellular matrix proteins.

Persistent vascular congestion in male spontaneously hypertensive rats contributes to delayed recovery of renal function following renal ischemia perfusion compared with females.

Acute kidney injury (AKI) due to ischemia is a serious and frequent clinical complication with mortality rates as high as 80%. Vascular congestion in the renal outer medulla occurs early after ischemia reperfusion (IR) injury, and congestion has been linked to worsened outcomes following IR. There is evidence implicating both male sex and preexisting hypertension as risk factors for poor outcomes following IR. The present study tested the hypothesis that male spontaneously hypertensive rats (SHR) have greater vascular congestion and impaired renal recovery following renal IR vs. female SHR and normotensive male Sprague-Dawley rats (SD). Thirteen-week-old male and female SHR and SD were subjected to sham surgery or 30 min of warm bilateral ischemia followed by reperfusion. Rats were euthanized 24 h or 7 days post-IR. IR increased renal injury in all groups vs. sham controls at 24 h. At 7 days post-IR, injury remained elevated only in male SHR. Histological examination of SD and SHR kidneys 24 h post-IR showed vascular congestion in males and females. Vascular congestion was sustained only in male SHR 7 days post-IR. To assess the role of vascular congestion on impaired recovery following IR, additional male and female SHR were pretreated with heparin (200 U/kg) prior to IR. Heparin pretreatment reduced IR-induced vascular congestion and improved renal function in male SHR 7 days post-IR. Interestingly, preventing increases in blood pressure (BP) in male SHR did not alter sustained vascular congestion. Our data demonstrate that IR-induced vascular congestion is a major driving factor for impaired renal recovery in male SHR.

Sacubitril/Valsartan Improves Sexual Function and Fibrosis of the Clitoral and Vaginal Tissues in Female Spontaneously Hypertensive Rats.

:Female sexual dysfunction is common in hypertension. The effects of sacubitril/valsartan (SAC/VAL) as a potential therapy for hypertension and heart failure have not been studied in relation to sexual function and genital fibrosis in female spontaneously hypertensive rats (SHRs). Thirty female SHRs were administered VAL, SAC/VAL, or saline. Ten normotensive female Wistar–Kyoto (WKY) rats were included in the control group. We assessed estrous cyclicity and sexual behavior in the female rats. In addition, the morphology of clitoral and vaginal tissues was evaluated by histological analyses. Western blotting and enzyme-linked immunosorbent assays were used to assess the levels of fibrotic markers in vaginal and clitoral tissues. Furthermore, the protein levels of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), phosphoinositide-3-kinase (PI3K), and AKT expression were measured by Western blotting. SAC/VAL treatment improved hypertension-induced sexual dysfunction, exhibited as a prolonged estrus phase, increased receptivity and proceptive events, and decreased aggressive events, compared with those of VAL treatment and control SHRs without treatments. In addition, SAC/VAL-treated SHRs had lower levels of fibrotic markers, estradiol, and estrogen receptor α/β than the levels of VAL-treated SHRs or SHRs without treatment. Moreover, SAC/VAL decreased p-PTEN expression and increased p-PI3K and p-AKT expression at the protein level compared with those in VAL treatment alone. VAL and SAC/VAL treatments have significantly increased sexual receptivity and proceptivity, decreased aggressiveness, and improved the fibrosis of vaginal and clitoral tissues in female SHRs. However, SAC/VAL treatment shows more effective results compared with VAL treatment, which may be related to the PTEN/PI3K/AKT pathway.