Stroke-prone Spontaneously Hypertensive Rats Research Articles

Activation of NLRP3 inflammasome in a rat model of cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is increasingly being recognized as a leading contributor to cognitive impairment in the elderly. However, there is a lack of effective preventative or therapeutic options for CSVD. In this exploratory study, we investigated the interplay between neuroinflammation and CSVD pathogenesis as well as the cognitive performance, focusing on NLRP3 signaling as a new therapeutic target. Spontaneously hypertensive stroke-prone (SHRSP) rats served as a CSVD model. We found that SHRSP rats showed decline in learning and memory abilities using morris water maze test. Activated NLRP3 signaling and an increased expression of the downstream pro-inflammatory factors, including IL (interleukin)-6 and tumor necrosis factor α were determined. We also observed a remarkable increase in the production of pyroptosis executive protein gasdermin D, and elevated astrocytic and microglial activation. In addition, we identify several neuropathological hallmarks of CSVD, including blood-brain barrier breakdown, white matter damage, and endothelial dysfunction. These results were in correlation with the activation of NLRP3 inflammasome. Thus, our findings reveal that the NLRP3-mediated inflammatory pathway could play a central role in the pathogenesis of CSVD, presenting a novel target for potential CSVD treatment.

Pregnancy-associated changes in urinary uromodulin excretion in chronic hypertension.

Pregnancy involves major adaptations in renal haemodynamics, tubular, and endocrine functions. Hypertensive disorders of pregnancy are a leading cause of maternal mortality and morbidity. Uromodulin is a nephron-derived protein that is associated with hypertension and kidney diseases. Here we study the role of urinary uromodulin excretion in hypertensive pregnancy. Urinary uromodulin was measured by ELISA in 146 pregnant women with treated chronic hypertension (n = 118) and controls (n = 28). We studied non-pregnant and pregnant Wistar Kyoto and Stroke Prone Spontaneously Hypertensive rats (n = 8/strain), among which a group of pregnant Stroke-Prone Spontaneously Hypertensive rats was treated with either nifedipine (n = 7) or propranolol (n = 8). In pregnant women, diagnosis of chronic hypertension, increased maternal body mass index, Black maternal ethnicity and elevated systolic blood pressure at the first antenatal visit were significantly associated with a lower urinary uromodulin-to-creatinine ratio. In rodents, pre-pregnancy urinary uromodulin excretion was twofold lower in Stroke-Prone Spontaneously Hypertensive rats than in Wistar Kyoto rats. During pregnancy, the urinary uromodulin excretion rate gradually decreased in Wistar Kyoto rats (a twofold decrease), whereas a 1.5-fold increase was observed in Stroke-Prone Spontaneously Hypertensive rats compared to pre-pregnancy levels. Changes in uromodulin were attributed by kidney injury in pregnant rats. Neither antihypertensive changed urinary uromodulin excretion rate in pregnant Stroke-Prone Spontaneously Hypertensive rats. In summary, we demonstrate pregnancy-associated differences in urinary uromodulin: creatinine ratio and uromodulin excretion rate between chronic hypertensive and normotensive pregnancies. Further research is needed to fully understand uromodulin physiology in human pregnancy and establish uromodulin's potential as a biomarker for renal adaptation and renal function in pregnancy.

Theobromine improves hyperactivity, inattention, and working memory via modulation of dopaminergic neural function in the frontal cortex of spontaneously hypertensive rats.

Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder and dopaminergic dysfunction in the prefrontal cortex (PFC) may play a role. Our previous research indicated that theobromine (TB), a methylxanthine, enhances cognitive function in rodents via the PFC. This study investigates TB's effects on hyperactivity and cognitive function in stroke-prone spontaneously hypertensive rats (SHR), an ADHD animal model. Male SHRs (6-week old) received a diet containing 0.05% TB for 40 days, while control rats received normal diets. Age-matched male Wistar-Kyoto rats (WKY) served as genetic controls. During the TB administration period, we conducted open-field tests and Y-maze tasks to evaluate hyperactivity and cognitive function, then assessed dopamine concentrations and tyrosine hydroxylase (TH), dopamine receptor D1-5 (DRD1-5), dopamine transporter (DAT), vesicular monoamine transporter-2 (VMAT-2), synaptosome-associated protein-25 (SNAP-25), and brain-derived neurotrophic factor (BDNF) expressions in the PFC. Additionally, the binding affinity of TB for the adenosine receptors (ARs) was evaluated. Compared to WKY, SHR exhibited hyperactivity, inattention and working memory deficits. However, chronic TB administration significantly improved these ADHD-like behaviors in SHR. TB administration also normalized dopamine concentrations and expression levels of TH, DRD2, DRD4, SNAP-25, and BDNF in the PFC of SHR. No changes were observed in DRD1, DRD3, DRD5, DAT, and VMAT-2 expression between SHR and WKY rats, and TB intake had minimal effects. TB was found to have affinity binding to ARs. These results indicate that long-term TB supplementation mitigates hyperactivity, inattention and cognitive deficits in SHR by modulating dopaminergic nervous function and BDNF levels in the PFC, representing a potential adjunctive treatment for ADHD.

Persistent brain exposure to high sodium induces stroke onset by upregulation of cerebral microbleeds and oxidative stress in hypertensive rats.

High salt intake induces hypertension and enhances stroke onset. However, whether an increase in brain sodium exposure itself is harmful and has poor prognosis remains unknown. Therefore, we employed hypertensive rats that underwent intracerebroventricular (ICV) infusion of sodium for 28 days and evaluated stroke onset and related cytotoxic brain injuries. Forty-seven spontaneously hypertensive stroke-prone (SHRSP) and 39 normotensive rats (Wistar Kyoto rats [WKY]) underwent persistent ICV infusion of the following four solutions: artificial cerebrospinal fluid, 0.9%, 2.7%, and 9% saline for 28 days. We evaluated stroke onset and all-cause mortality between SHRSP and WKY at each ICV sodium concentration as the primary endpoints. Our secondary objective was to explore histological brain injuries associated with SHRSP induced by high sodium ICV. The results indicated that ICV infusion of 2.7% and 9% sodium showed a significant increase in stroke onset, decrease in body weight, and increase rate of brain water content in SHRSP compared to WKY. Increased blood pressure was not observed for ICV infusion of high sodium, while serum sodium concentration was significantly increased in SHRSP compared to WKY. Histological evaluations revealed that higher sodium infusion significantly increased the number of activated microglia, superoxide, neuronal cell loss, and microbleeds compared to WKY and SHRSP with 0.9% sodium. We conclude that persistent exposure to high sodium in the brain is one of the risk factors for stroke onset upregulating cerebral microbleeds and oxidative stress in hypertensive rats.

Polygenic genetic variation affecting antibody formation underlies hypertensive renal injury in the stroke-prone spontaneously hypertensive rat.

During development of the spontaneously hypertensive rat (SHR), several distinct but closely related lines were generated. Most lines are resistant to hypertensive renal disease. However, the SHR-A3 line (stroke-prone SHR) experiences end-organ injury (EOI) and provides a model of injury susceptibility that can be used to uncover genetic causation. In the present study, we generated a congenic line in which three distinct disease loci in SHR-A3 are concurrently replaced with homologous loci from an injury-resistant SHR line (SHR-B2). Verification that all three loci were homozygously replaced in this triple congenic line [SHR-A3(Trip B2)] while the genetic background of SHR-A3 was fully retained was obtained by whole genome sequencing. Congenic genome substitution was without effect on systolic blood pressure [198.9 ± 3.34 mmHg, mean ± SE, SHR-A3(Trip B2) = 194.7 ± 2.55 mmHg]. Measures of renal injury (albuminuria, histological injury scores, and urinary biomarker levels) were reduced in SHR-A3(Trip B2) animals, even though only 4.5 Mbases of the 2.8 Gbases of the SHR-B2 genome (0.16% of the genome) was transferred into the congenic line. The gene content of the three congenic loci and the functional effects of gene polymorphism within suggest a role of immunoglobulin in EOI pathogenesis. To prove the role of antibodies in EOI in SHR-A3, we generated an SHR-A3 line in which expression from the immunoglobulin heavy chain gene was knocked out (SHR-A3-IGHKO). Animals in the SHR-A3-IGHKO line lack B cells and immunoglobulin, but the hypertensive phenotype is not affected. Renal injury, however, was reduced in this line, confirming a pathogenic role for immunoglobulin in hypertensive EOI in this model of heritable risk.NEW & NOTEWORTHY Here, we used a polygenic animal model of hypertensive renal disease to show that genetic variation affecting antibody formation underlies hypertensive renal disease. We proved the genetic thesis by generating an immunoglobulin knockout in the susceptible animal model.

Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.

Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR). We used Dkk3-/- mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR. Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the central nervous system (CNS). Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke. These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt (protein kinase B)/eNOS hypotensive axis.

S-22-7: GENOMIC ANALYSES OF HYPERTENSION-ASSOCIATED DISEASES ACROSS ETHNIC GROUPS AND SPECIES

To explore genetic architecture of hypertension and end-organ damages, we performed integrative genomic analyses in both humans and inbred hypertensive rat strains. In humans, noting that hypertension is more prevalent and contributes to the increased risk of stroke in people of East Asian ancestry, we performed a multi-stage GWAS for blood pressure (BP) principally in East Asians and meta-analysis in East Asians and Europeans. We reported 19 new genetic loci and ancestry-specific BP variants, conforming to a common ancestry-specific variant association model. Some of the ancestry-specific association signals are influenced by a selective sweep. These data provide new evidence for the role of common ancestry-specific variants and natural selection in ethnic differences in complex traits such as BP. In rats, we also performed genomic analyses of BP and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP). By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n = 1415) and fine congenic mapping (maximum n = 8704) plus comparative analysis with human, transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY). We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this analysis, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein-kinin/renin-angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. Furthermore, to unravel the mechanisms underlying antihypertensive drug-induced amelioration of end-organ damages, we performed transcriptomic analysis on the heart and the kidney, with administration of antihypertensive drugs to two inbred rat strains (SHRSP and WKY). Noticeable changes of mRNA expression were induced particularly by RAS blockade, for example, for genes in the natriuretic peptide system (Nppb and Corin) in the heart and for those in the KKS/RAS (Ren and rat Klk1 paralogs) and those related to calcium ion binding (Calb1 and Slc8a1) in the kidney. Taken together, we provide genetic epidemiological evidence for ethnic differences in hypertension susceptibility and also in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, likely across species.

PS-B03-7: MAGNESIUM SULPHATE TREATMENT DURING HYPERTENSIVE PREGNANCY IMPROVES MATERNAL BUT NOT FETAL OUTCOMES IN RATS

Objective: There has been a recent rise in the incidence of cardiovascular disease amongst women of child-bearing age and hypertensive disorders in pregnancy, in particular pre-eclampsia. Though it is frequently diagnosed, therapeutic interventions are lacking. Magnesium sulphate (MgSO4) is known to induce a vasodilatory effect via increased NO production and improved endothelial function and is currently used to prevent the progression to eclampsia and seizures from pre-eclampsia in the clinic. Magnesium homeostasis is also crucial during a healthy pregnancy and hypomagnesemia has been linked to cardiovascular disease. We hypothesised that MgSO4 may be used as a preventative therapeutic during hypertensive pregnancies in a rodent model of super-imposed pre-eclampsia. Design and Methods: Spontaneously hypertensive stroke prone (SHRSP) female rats were time mated. On gestational day (GD) 10.5 0.9% saline (SHAM, n = 10) or 750ng/kg/min of ANGII (n = 17) were administered via mini pump. A subset of ANGII and SHAM animals received daily 1% w/v MgSO4 drinking water from GD0.5 (n = 4/group; SHAM/ANGII+MgSO4). Blood pressure, cardiac function, and uteroplacental blood flow were measured by tail-cuff plethysmography and Doppler ultrasound pre-pregnancy (PP) and at GD6.5, 12.5, and 18.5. Urine samples were collected by metabolic cage at PP, GD6.5, and GD18.5 for measurement of proteinuria. Fetal and placental measurements were collected at sacrifice. Results: ANGII shows features consistent with SPE in humans via decreased stroke volume and elevated blood pressure alongside decreased fetal and placental weights vs SHAM (****p < 0.0001, **p < 0.01, *p < 0.05). Additionally, ANGII resulted in significantly altered fluid homeostasis and proteinuria vs SHAM (****p < 0.0001, ***p < 0.001, **p < 0.01). ANGII+MgSO4 showed a decrease in blood pressure vs ANGII (**p < 0.01). Treatment with MgSO4 had no significant effect on cardiac function, though there was a trend towards improvement in delta cardiac output. Neither ANGII nor additional MgSO4 altered indices of uteroplacental flow vs ANGII or SHAM. ANGII+MgSO4 exhibited a significant reduction in proteinuria vs ANGII (*p < 0.05) as well as improvements in fluid homeostasis. Both fetal and placental weights were significantly reduced in ANGII+MgSO4 vs SHAM (**p < 0.001) indicating a detrimental effect. Conclusions: These results suggest 1% w/v MgSO4 may be beneficial as a preventative therapeutic in pregnancies affected by super-imposed pre-eclampsia for maternal but not fetal outcomes. Further work is needed to elucidate the effects of MgSO4 in the context of hypertensive pregnancy.

Stroke-Prone SHR as Experimental Models for Cardiovascular Disease Risk Reduction in Humans.

Since stroke-prone spontaneously hypertensive rats (SHRSP) develop hypertension and stroke without exception, the prevention or reduction of risk by various nutrients was tested on blood pressure and the mortality caused by stroke and cardiovascular diseases (CVD). In addition to sodium (Na) accelerating hypertension and stroke and potassium (K) counteracting the adverse effect of Na, taurine (Tau), rich in seafood, and magnesium (Mg) contained in soy, nuts, grains, etc., were proven to reduce stroke and CVD and improve survival. Therefore, the Cardiovascular Diseases and Alimentary Comparison Study was started in 1985 to explore the association of biomarkers of diet in 24 h urine (24U) with CVD risks, and about 100 males and 100 females aged 48-56 in each of 50 populations were studied until 1995. Linear regression analysis indicated that the 24U Tau/creatinine and Mg/creatinine ratios were inversely associated with body mass index, systolic and diastolic blood pressure, and total cholesterol. In comparison with six Euro-Western regions, 24U Tau and Mg collected from six regions, respectively, in Japan and the Mediterranean countries were significantly higher and were significantly associated with lower CVD risks. Diets rich in Tau and Mg were concluded to be contributory to the prevention of CVD in SHRSP and humans.

Distinct Brain Proteomic Signatures in Cerebral Small Vessel Disease Rat Models of Hypertension and Cerebral Amyloid Angiopathy.

Cerebral small vessel diseases (CSVDs) are prominent contributors to vascular cognitive impairment and dementia and can arise from a range of etiologies. Cerebral amyloid angiopathy (CAA) and hypertension (HTN), both prevalent in the elderly population, lead to cerebral microhemorrhages, macrohemorrhages, and white matter damage. However, their respective underlying mechanisms and molecular events are poorly understood. Here, we show that the transgenic rat model of CAA type 1 (rTg-DI) exhibits perivascular inflammation that is lacking in the spontaneously hypertensive stroke-prone (SHR-SP) rat model of HTN. Alternatively, SHR-SP rats display notable dilation of arteriolar perivascular spaces. Comparative proteomics analysis revealed few shared altered proteins, with key proteins such as ANXA3, H2A, and HTRA1 unique to rTg-DI rats, and Nt5e, Flot-1 and Flot-2 unique to SHR-SP rats. Immunolabeling confirmed that upregulation of ANXA3, HTRA1, and neutrophil extracellular trap proteins were distinctly associated with rTg-DI rats. Pathway analysis predicted activation of TGF-β1 and TNFα in rTg-DI rat brain, while insulin signaling was reduced in the SHR-SP rat brain. Thus, we report divergent protein signatures associated with distinct cerebral vessel pathologies in the SHR-SP and rTg-DI rat models and provide new mechanistic insight into these different forms of CSVD.

Hypertension-Associated Genes in the Mesenteric Artery of Three Spontaneously Hypertensive Rat Substrains Identified Using a DNA Array Method

Although the mesenteric artery plays a key role in regulating peripheral blood pressure, the molecular mechanisms that underlie the development of essential hypertension are not yet fully understood. We explored candidate genes for hypertension using three related strains of spontaneously hypertensive rats (SHRs) that mimic human essential hypertension. In this study we used DNA microarrays, a powerful tool for studying genetic diseases, to compare gene expression in the mesenteric artery of three SHR substrains: SHR, stroke-prone SHR (SHRSP), and malignant SHRSP (M-SHRSP). Compared to normotensive 6-week old Wistar Kyoto rats (WKY), higher blood pressure correlated with overexpression of 31 genes and with down regulation of 24 genes. Adam23, which negatively regulates potassium current, and the potassium channel genes, Kcnc2 and Kcnq5, were associated with the onset of hypertension. In addition, Spock2 and Agtrap were identified as strengtheners of hypertension by analyzing up and down regulated genes at 9-weeks of age. Adam23, Kcnc2 and Kcnq5 appear to be factors for the onset of hypertension, while Spock2 and Agtrap are as factors that strengthen hypertension. These findings contribute to our understanding of the pathophysiology of hypertension and to the development of treatment for this condition.

Characterization of perivascular space pathology in a rat model of cerebral small vessel disease by in vivo magnetic resonance imaging

One of the most common causes of dementia is cerebral small vessel disease (SVD), which is associated with enlarged perivascular spaces (PVS). Clinically, PVS are visible as hyperintensities on T2-weighted (T2w) magnetic resonance images (MRI). While rodent SVD models exhibit arteriolosclerosis, PVS have not been robustly documented by MRI casting doubts on their clinical relevance. Here we established that the severity of SVD in spontaneously hypertensive stroke prone (SHRSP) rats correlated to ‘moderate’ SVD in human post-mortem tissue. We then developed two approaches for detecting PVS in SHRSP rats: 1) T2w imaging and 2) T1-weighted imaging with administration of gadoteric acid into cerebrospinal fluid. We applied the two protocols to six Wistar-Kyoto (WKY) control rats and thirteen SHRSP rats at ∼12 month of age. The primary endpoint was the number of hyperintense lesions. We found more hyperintensities on T2w MRI in the SHRSP compared to WKY rats (p-value = 0.023). CSF enhancement with gadoteric acid increased the visibility of PVS-like lesions in SHRSP rats. In some of the SHRSP rats, the MRI hyperintensities corresponded to enlarged PVS on histopathology. The finding of PVS-like hyperintensities on T2w MRI support the SHRSP rat’s clinical relevance for studying the underlying pathophysiology of SVD.

Impact of Non-pharmacological Chronic Hypertension on a Transgenic Rat Model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA), a common comorbidity of Alzheimer’s disease (AD), is a cerebral small vessel disease (CSVD) characterized by deposition of fibrillar amyloid β (Aβ) in blood vessels of the brain and promotes neuroinflammation and vascular cognitive impairment and dementia (VCID). Hypertension, a prominent non-amyloidal CSVD, has been found to increase risk of dementia, but clinical data regarding its effects in CAA patients is controversial. To understand the effects of hypertension on CAA, we bred rTg-DI transgenic rats, a model of CAA, with spontaneously hypertensive, stroke prone (SHR-SP) rats producing bigenic rTg-DI/SHR-SP and non-transgenic SHR-SP littermates. At 7 months (M) of age, cohorts of both rTg-DI/SHR-SP and SHR-SP littermates exhibit elevated systolic blood pressures. However, transgene human amyloid β-protein (Aβ) precursor and Aβ peptide levels, as well as behavioral testing showed no changes between bigenic rTg-DI/SHR-SP and rTg-DI rats. Subsequent cohorts of rats were aged further to 10 M where bigenic rTg-DI/SHR-SP and SHR-SP littermates exhibit elevated systolic and diastolic blood pressures. Vascular amyloid load in hippocampus and thalamus was significantly decreased, whereas pial surface vessel amyloid increased, in bigenic rTg-DI/SHR-SP rats compared to rTg-DI rats suggesting a redistribution of vascular amyloid in bigenic animals. There was activation of both astrocytes and microglia in rTg-DI rats and bigenic rTg-DI/SHR-SP rats not observed in SHR-SP rats indicating that glial activation was likely in response to the presence of vascular amyloid. Thalamic microbleeds were present in both rTg-DI rats and bigenic rTg-DI/SHR-SP rats. Although the number of thalamic small vessel occlusions were not different between rTg-DI and bigenic rTg-DI/SHR-SP rats, a significant difference in occlusion size and distribution in the thalamus was found. Proteomic analysis of cortical tissue indicated that bigenic rTg-DI/SHR-SP rats largely adopt features of the rTg-DI rats with enhancement of certain changes. Our findings indicate that at 10 M of age non-pharmacological hypertension in rTg-DI rats causes a redistribution of vascular amyloid and significantly alters the size and distribution of thalamic occluded vessels. In addition, our findings indicate that bigenic rTg-DI/SHR-SP rats provide a non-pharmacological model to further study hypertension and CAA as co-morbidities for CSVD and VCID.

Abstract P232: Pregnancy Induced Changes In Urinary Uromodulin Excretion In Normotensive And Chronic Hypertensive Rodent Models

Hypertensive disorders complicate 1-4% of pregnancies and are the leading cause of maternal death. Pregnancy involves major adaptions in renal hemodynamics, tubular and endocrine functions. Uromodulin (UMOD) is a renal protein that plays a role in renal sodium and divalent cation transport and is associated with hypertension and kidney diseases. We aimed to understand the role of blood pressure (BP) in urinary UMOD excretion during pregnancy. Pregnant Wistar Kyoto (WKY) and Stroke Prone Spontaneously Hypertensive (SHRSP) rats (n=8) were studied for 18.5 gestational days (GD). A group of pregnant SHRSP were treated with the beta-blocker propranolol (100mg/kgBW/day; n=8) and the calcium channel blocker nifedipine (25mg/kgBW/day; n=7) from GD 0.5. Systolic BP (SBP; tail cuff plethysmography), urine and plasma characteristic were monitored at baseline, GD3.5, GD12.5 and GD 18.5. UMOD concentration were measured in 24hr urine with ELISA. Pregnant SHRSP were hypertensive throughout the gestation compared to WKY (delta SBP 22.6 ± 3.6 mmHg, p<0.0001). At baseline (pre-pregnancy), urinary UMOD levels were 2-fold higher in WKY compared to SHRSP (p<0.05). During pregnancy, the urinary UMOD gradually decreased in WKY (2-fold decrease, p<0.05), whereas a 1.5-fold increase (p<0.05) was observed in hypertensive SHRSP compared to pre-pregnancy. The changes observed in urinary levels corresponded to total kidney UMOD levels at GD18.5, wherein SHRSP kidney shows 1.3-fold (p<0.01) greater expression of UMOD compared to WKY. Nifedipine reduced BP in pregnant SHRSP (delta SBP between GD18.5 and baseline: 44.3 ± 7.4 mmHg, p<0.0001) while propranolol did not affect BP. However, neither nifedipine nor propranolol changed urinary UMOD excretion in pregnant SHRSP. BP was not significantly correlated with urinary uromodulin levels in pregnant WKY (Pearson, r=-0.01, p 0.95) and SHRSP without (r=-0.16, p 0.46) or with (nifedipine r=0.26, p 0.31; propranolol r=0.17, p 0.45) antihypertensive treatment. We demonstrate differences in UMOD excretion between hypertensive and normotensive pregnancy and changes during pregnancy that are at least in part BP independent. Our data provide novel insights into molecular changes associated with hypertensive pregnancy.

Kudzu isoflavone C‐glycosides: Analysis, biological activities, and metabolism

AbstractRadix Pueraria (the root of kudzu Pueraria lobota) is a popular traditional Chinese medicine used in dietary supplements in Western markets and has potential health benefits. Kudzu roots are rich in isoflavones C‐ and O‐glycosides, of which puerarin (daidzein 8‐C‐glucoside) is the most abundant isoflavone. Puerarin is a unique isoflavone that it is resistant to intestinal hydrolysis and has a wide range of effects in preventing metabolic diseases. Our previous studies indicate that chronic exposure to a diet enriched in puerarin significantly reduces serum total cholesterol, arterial blood pressure, insulin resistance and hyperglycemia in ovariectomized, stroke‐prone spontaneously hypertensive rats (SP‐SHR), a model of metabolic syndrome. Further, our studies demonstrate that puerarin is absorbed as the intact glucoside and acutely improves glucose tolerance, indicating that it has potential for the prevention and treatment of diabetes. This paper reviews recent progress in the understanding of biological activities and metabolism and in the analysis of puerarin in kudzu root extracts or supplements.

Reduced Bile Acid Signaling Contributes to Hypertension Development in Spontaneously Hypertensive Stroke‐Prone Rats

Exciting recent findings have identified that gut microbiota plays an essential role in blood pressure (BP) regulation. Studies from our lab and others have shown a causal role of gut dysbiosis in hypertension development in the spontaneously hypertensive stroke-prone (SHRSP) rat and several other animal models. One key mechanism by which the microbiota influences the host is through the generation/modification of metabolites. Among these metabolites, bile acids (BAs), are derived from the liver and modified by gut bacteria. Many BA species cross the gut epithelial barrier into the host circulation and influence inflammation, metabolism, and vascular function through the binding and activation of BA receptors, including TGR5. We hypothesized that disrupted BA signaling, as a result of gut dysbiosis, contributes to the development of hypertension. We observed a significant reduction of several BAs in systemic plasma of SHRSP as compared to WKY, including the TGR5 agonists, cholic acid (CA), hyocholic acid (HCA), hyodeoxycholic acid (HDCA), and tauro-lithocholic acid (TLCA) (p<.05). We next supplemented WKY and SHRSP rats with 0.5% CA in the diet and measured systolic blood pressure (SBP) bi-weekly. After 15 weeks of treatment, systemic plasma was collected for BA analysis and endothelium-dependent dilation was assessed in the isolated aorta. We observed that CA supplementation significantly increased CA, HCA, HDCA, and TLCA in systemic plasma of SHRSP (p<.0001). CA supplementation also significantly reduced SBP of SHRSPs, with an average decrease of 23 mmHg over the final 6 weeks of treatment (p<.05) compared to SHRSP controls. Chronic CA treatment also significantly improved vasodilatory response to increasing doses of acetylcholine (ACh) in the isolated aorta of SHRSPs (p<.05). Next, we determined if BAs had their beneficial effects through activation of the BA receptor TGR5. Beginning at 6 weeks of age, SHRSPs were randomized into vehicle control or oleanolic acid (OA, 10 mg/kg IP daily), a selective TGR5 agonist, treatment groups. OA significantly decreased SBP by an average of 15 mmHg over the final 6 weeks of the treatment, compared to controls (p<.05). Furthermore, 15 weeks of OA treatment significantly improved vasodilatory response to increasing doses of ACh in isolated mesenteric arteries of SHRSPs (p<.05). Because systemic and neuronal inflammation also contributes to the development of hypertension, we examined the effects of TGR5 activation by OA treatment on inflammation in the gut and brain of SHRSPs. Using flow cytometry, we determined that OA treatment increased the percentage of anti-inflammatory Tregs and decreased pro-inflammatory T helper 17 cells in both cecum and brain of SHRSP (p<.05). OA treatment also significantly reduced interferon-γ+ T cells in the cecum of SHRSP. These data suggest an anti-inflammatory role of TGR5 signaling in the gut and brain. We conclude that reduced BA-mediated TGR5 signaling contributes to the development of hypertension in SHRSPs, and that activating TGR5 may be a potential therapeutic approach to attenuate vascular endothelial dysfunction and associated hypertension.

Integrative genomic analysis of blood pressure and related phenotypes in rats.

ABSTRACTDespite remarkable progress made in human genome-wide association studies, there remains a substantial gap between statistical evidence for genetic associations and functional comprehension of the underlying mechanisms governing these associations. As a means of bridging this gap, we performed genomic analysis of blood pressure (BP) and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP), both of which are unique genetic models of severe hypertension and cardiovascular complications. By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n=1415), fine congenic mapping (maximum n=8704), pharmacological intervention and comparative analysis with transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY); e.g. inter-strain BP differences between SHRSP and WKY could be largely explained by an aggregate of BP changes in seven SHRSP-derived consomic strains. We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this study, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein–kinin/renin–angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. In combination with a pharmacological intervention, we provide in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, in a rat polygenic hypertension model.

Left-ventricular hypertrophy in 18-month-old donor rat hearts was not associated with graft dysfunction in the early phase of reperfusion after cardiac transplantation\u2013gene expression profiling

The use of hearts with left-ventricular (LV) hypertrophy (LVH) could offer an opportunity to extend the donor pool for cardiac transplantation. We assessed the effects of LVH in 18-month-old spontaneously hypertensive stroke-prone (SHRSP) donor rats and following transplantation. In donors, cardiac function and structural alterations were assessed. Then, the hearts were transplanted into young normotensive-rats. We evaluated LV graft function 1 h after transplantation. The myocardial expression of 92 genes involved in apoptosis, inflammation, and oxidative-stress was profiled using PCR-array. Compared to controls, SHRSP-rats developed LVH, had increased LV systolic performance (slope of the end-diastolic pressure-volume (PV) relationship: 1.6±0.2 vs 0.8±0.1mmHg/μl, p<0.05) accompanied by diastolic dysfunction [prolonged time constant of LV pressure decay (Tau: 15.8±0.6 vs 12.3±0.5ms) and augmented diastolic stiffness (LV end-diastolic PV relationship: 0.103±0.012 vs 0.045±0.006mmHg/ml), p<0.05]. They presented ECG changes, myocardial fibrosis, and increased nitrotyrosine immunoreactivity and plasma troponin-T and creatine kinase-CM levels. After transplantation, even though the graft contractility was better in SHRSP rats compared to controls, the adverse impact of ischemia/reperfusion-injury on contractility was not altered (Ees ratio after versus before transplantation: 32% vs 29%, p>0.05). Whereas nitrotyrosine immunoreactivity was higher, myeloperoxidase-positive cell infiltration was decreased in the SHRSP+transplanted compared to control+transplanted. Among the tested genes, LVH was associated with altered expression of 38 genes in donors, while transplantation of these hearts resulted in the change of four genes. Alterations in 18-month-old donor hearts, as a consequence of hypertension and LVH, were not associated with graft dysfunction in the early phase of reperfusion after transplantation.

Pharmacological characterization of MT-1207, a novel multitarget antihypertensive agent.

Hypertension is a serious public health problem worldwide. MT-1207, chemically named 3-(4-(4-(1H-benzotriazole-1-yl)butyl)piperazine-1-yl) benzisothiazole hydrochloride, is a new chemical entity that has entered into clinical trial as antihypertensive agent in China. In this paper we report the pharmacological profile of MT-1207 regarding its acute, subacute, and long-term effects on hypertensive animal models, and its actions on isolated organs in vitro as well as its molecular targets. Blood pressure (BP) was measured in conscious animals; amlodipine was taken as a positive control drug. We showed that both single dose of MT-1207 (1.25-20 mg/kg, ig) in spontaneously hypertensive rats (SHR) and MT-1207 (0.25-6 mg/kg, ig) in two-kidney one-clip (2K1C) dogs dose-dependently decreased BP. MT-1207 quickly decreased BP within 5 min after administration; the hypotensive effect lasted for 8 and 12 h, respectively, in SHR and 2K1C dogs without reflex increase in heart rate. Multiple doses of MT-1207 (5 mg · kg-1 · d-1 in SHR; 2 mg · kg-1 · d-1 in 2K1C dogs, for 7 days) significantly decreased BP, slightly reduced heart rate, and both of them recovered after withdrawal. Long-term administration of MT-1207 (10 mg · kg-1 · d-1 for 4 months or more time) produced a stable BP reduction, improved baroreflex sensitivity, reduced renal and cardiovascular damage in SHR, and delayed stroke occurrence and death in stroke-prone SHR. In isolated rat aortic rings precontracted by adrenaline, KCl, noradrenaline or 5-hydroxytryptamine (5-HT), MT-1207 (10-9-10-4 M) caused concentration-dependent relaxation. In a panel of enzyme activity or radioligand binding assays of 87 molecular targets, MT-1207 potently inhibited adrenergic α1A, α1B, α1D, and 5-HT2A receptors with Ki < 1 nM. The antagonism of MT-1207 against these receptors was confirmed in isolated rabbit arteries. We conclude that MT-1207 is a novel and promising single-molecule multitarget agent for hypertension treatment to reduce hypertensive organ damage and stroke mortality.

Abstract P52: Temporal Progression of Cerebral Small Vessel Disease Lesions on Brain Magnetic Resonance Imaging in Spontaneously Hypertensive Stroke Prone Rats

Introduction: Spontaneously Hypertensive Rats- Stroke Prone (SHRSP) are a relevant model for human cerebral small vessel disease (cSVD). However, data are still lacking regarding the neuroimaging correlates of cSVD lesions in SHRSP and their temporal evolution in relationship to histological findings. Methods: 40 SHRSP and 40 Wistar Kyoto control (WKY) male rats were divided into 4 groups (10 WKY and 10 SHRSP) per group. Systolic blood pressure (SBP) was measured using tail-cuff device, weekly. Select animals of each group had brain MRI at 9.4T machine at 7, 16, 24, and 32 weeks of age. Volumetric analysis was completed using manual segmentation of the total brain, ventricles and bilateral hippocampi. Following MRI, brain histology was completed at the same time points. Results: At 6 weeks, SBP was similar (WKY 123.8±0.1 vs SHRSP 130.7±3.2, P=0.09). SHRSP developed hypertension between 9-11 weeks of age and maintained it throughout the experiment (SBP at 31 weeks: WKY 138.1±6 vs SHRSP 169.1±6.7, p=0.0006). At 7 weeks, brain MRI was normal in SHRSP and WKY. Histology was largely unremarkable except for few areas of red blood cell extravasation in couple of SHRSP. At 16 weeks, MRI was normal in WKY and it showed small subcortical hyperintensity on T2 sequences in one SHRSP while histology showed microbleeds (MBs) in 85% of SHRSP. At 24 weeks, brain MRI consistently identified subcortical hyperintensities in SHRSP and H&amp;E showed MBs in all SHRSP in addition to hemosiderin deposition and arteriosclerosis. LFB staining showed areas of demyelination in the corpus callosum. At 32 weeks, SHRSP had hydrocephalus and H&amp;E showed widespread hemosiderin deposition. Volumetric analysis showed larger ventricles in SHRSP (SHRSP 42.3±18.1 ml vs WKY 28.4±7.2 ml, p=0.013) and ventricle size significantly increased with age in SHRSP. Hippocampal and brain volumes were similar in both groups (hippocampal volume: WKY 84.5±8.1 ml vs SHRSP 81.6±2.6 ml, p=0.39; brain volume WKY 2103.4±100.4 ml vs SHRSP 2169.6 ±164.2 ml, p=0.2). Conclusions: SHRSP develop cSVD histological changes early in life and brain MRI showed consistent abnormalities at a later time point. These results have implications in defining cSVD phenotypes in SHRSP in future mechanistic studies.