Young WKY Research Articles

MiR-328 - a novel diagnostic and predictive atrial fibrillation biomarker in spontaneously hypertensive rats

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Romanian Ministry of Education and Research, CNCS-UEFISCDI Purpose Atrial fibrillation (AF) is the most common cardiac arrhythmia and a major cause of morbidity and mortality worldwide. To date, there are no reliable biomarkers for the diagnosis and prediction of AF. Alterations in the atrial expression of various microRNAs contributes to the pathogenesis of AF, suggesting that certain miRNAs may emerge as promising AF diagnostic and/or predictive biomarkers. We aimed to evaluate miR-328 as a potential biomarker for the prediction and diagnosis of AF in an experimental model of spontaneous AF in rats. Method Male spontaneously hypertensive rats (SHR) and their normotensive counterparts (WKY) were divided into 6 groups: young (12 weeks, n = 8 each), adult (26 weeks, n = 8 each), and elderly (46 weeks, n = 10 each) SHR and WKY. All animals were implanted with ECG radiotelemetry devices, the ECG was continuously monitored for 72 hours, and the number of spontaneous AF episodes was assessed. At the end of the protocol, blood and left atrium tissue samples were taken and miR-328 levels were quantified. miR-328 levels were normalized with U6 snRNA and compared between groups. Correlations between atrial and blood miR-328 levels, and between mir-328 levels and arrhythmic load were also assessed. Results None of the WKY rats and none of the young SHRs presented arrhythmic episodes; two of the adult SHRs and all elderly SHRs had spontaneous AF episodes. Young SHR and WKY rats had similar atrial miR-328 levels (p = 0.67). At adult age, atrial miR-328 levels were significantly higher in SHRs than in WKYs (1.20 ± 0.02 vs. 1.07 ± 0.04; p = 0.01). Between-groups differences were even higher in the elderly rats (1.59 ± 0.01 vs. 1.03 ± 0.03; p< 0.01). Among the SHRs, atrial miR-328 levels increased progressively with age (p = 0.02) and were strongly correlated with the circulating blood levels of miR-328 (r = 0.71; p = 0.04). Moreover, among the elderly SHRs, atrial miR-328 levels correlated positively with the number of arrhythmic episodes (r = 0.72; p< 0.01). Conclusions The present study demonstrates a strong association between increased miR-328 expression levels and the presence and burden of AF in the hypertensive rats, suggesting that miR-328 could be a promising biomarker for the diagnosis of AF. Moreover, our results demonstrate that in this model the increase in miR-328 levels precedes the onset of AF, suggesting that dynamic evaluation of miR-328 could be used to predict AF, at least in a hypertensive context. Further studies will have to validate these results in clinical settings and to assess miR-328 manipulation as a potential prophylactic and/or therapeutic strategy in AF.

Immunomodulatory Properties of Mesenchymal Stromal Cells Can Vary in Genetically Modified Rats.

Mesenchymal Stromal Cells (MSC) have been shown to exhibit immuno-modulatory and regenerative properties at sites of inflammation. In solid organ transplantation (SOT), administration of MSCs might lead to an alleviation of ischemia-reperfusion injury and a reduction of rejection episodes. Previous reports have suggested ‘MSC-preconditioning’ of macrophages to be partly responsible for the beneficial effects. Whether this results from direct cell-cell interactions (e.g., MSC trans-differentiation at sites of damage), or from paracrine mechanisms, remains unclear. Immunosuppressive capacities of MSCs from donors of different age and from genetically modified donor animals, often used for in-vivo experiments, have so far not been investigated. We conducted an in vitro study to compare paracrine effects of supernatants from MSCs extracted from young and old wild-type Wystar-Kyoto rats (WKY-wt), as well as young and old WKY donor rats positive for the expression of green fluorescent protein (WKY-GFP), on bone marrow derived macrophages (BMDM). Expression levels of Mannose receptor 1 (Mrc-1), Tumor necrosis factor α (TNFα), inducible NO synthase (iNos) and Interleukin-10 (IL-10) in BMDMs after treatment with different MSC supernatants were compared by performance of quantitative PCR. We observed different expression patterns of inflammatory markers within BMDMs, depending on age and genotype of origin for MSC supernatants. This must be taken into consideration for preclinical and clinical studies, for which MSCs will be used to treat transplant patients, aiming to mitigate inflammatory and allo-responses.

A1651 TAURINE IMPROVES REDUCED RENAL MEDULLARY BLOOD FLOW IN SPONTANEOUSLY HYPERTENSIVE RATS

Objectives: Present study was carried out to clarify the effect of renal nerve stimulation on renal cortical and medullary blood flow and the effects of chronic treatment of taurine on altered renal cortical and medullary blood flow distribution by renal nerve stimulation in spontaneously hypertensive rats (SHR). Methods: We used 6–7 (young) and 16–17 week old (adult) SHR and divided to taurine treated group and control group. Renal nerve surround left renal artery was stimulated electrically by 2, 5, 10 and 15 Hz under the anesthesia of isoflurane. Renal blood flow was measured using laser tissue flow meter using noncontact prove for cortical blood flow and contact glass-fiber needle type of prove for medullary flow. After a series of electrical stimulation, 1 mg of LNAME was injected and same protocol was repeated. Results: High frequency stimulation caused transient cortical blood flow elevation and which was followed by decrease of blood flow in young and adult WKY. In contrast, blood flow in renal medulla showed no significant changes compared with that in cortex. Young SHR showed a decrease of cortical and medullary blood flow by nerve stimulation. In adult SHP, blood flow reduction was similar to young SHR and medullary blood flow was more reduced than that in young rats. In adult WKY and young SHR, LNAME augmented the reduction of medullary blood flow by electrical stimulation. In adult SHR, LNAME showed no effects on blood flow reduction in medulla. Chronic treatment of taurine blunted the reduction of blood flow by electrical stimulation in medulla but not in cortex of adult SHR. Conclusion: Medullary blood flow is protected through the nitric oxide production in normotensive rats but this mechanism is blunted in SHR. Taurine could improve the reduction of renal medullary blood flow in aged SHR.

Prehypertensive juvenile spontaneously hypertensive rats have more orexin neurons than normotensive Wistar Kyoto rats

The neuropeptide orexin contributes to the regulation of blood pressure as part of its role in the control of arousal during wakefulness and motivated behavior. Recent work has suggested that an upregulated orexinergic system may contribute to the hypertensive phenotype of the SHR, with adult SHRs having significantly higher numbers of orexin neurons in the medial, but not lateral hypothalamus as compared to normotensive WKY and Wistar rats. (Clifford et al. 2015, Exp Physiol). However it is not known whether this upregulation is causing, or secondary to hypertensive pathologies seen in SHR periphery organs. As SHRs develop hypertension at 6 weeks of age, we sought to investigate the orexin system of prehypertensive SHRs. Aim: To determine the number of orexin neurons in young SHR and WKY rats before hypertension develops. Methods: Juvenile (3-5 weeks old) SHR (n = 8) and Wistar Kyoto (WKY, n = 8) male rats were euthanized, perfused and their brains sectioned and immunolabelled for orexin A. Labelled neurons were plotted and counted in the six densest hemisections (120 μm apart) of each brain. Results: There was a significantly higher number of orexin neurons (~ + 45%) in both the medial hypothalamus (medial to fornix) in hypertensive SHRs when compared to normotensive WKYs (119 ± 4 vs 86 ± 5, p < 0.0001 per hemisection) and the lateral hypothalamus (72 ± 3 vs 47 ± 3 per hemisection, p < 0.0001). Conclusion: These results show that the orexinergic system is highly upregulated in young SHRs well before hypertension develops, suggesting that hypertensive periphery develops in response to this increased sympathetic output.

Reduced carotid artery blood flow of spontaneously hypertensive rats is unaffected by aging from 20 weeks (young adulthood) to 65 weeks (upper middle age) (LB690)

The objective of this study was to quantify blood flow (using a perivascular flowprobe) and pressure (using a catheter‐tip transducer) in the common carotid artery (CCA) of anesthetized young adult (~20 week old) and upper middle aged (~65 week old) Wistar Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR). In young adult SHR vs. WKY, mean (172±10 vs. 82±2 mmHg), systolic (198±13 vs. 92±4), diastolic (151±9 vs. 72±5), and pulse pressure (48±5 vs. 20±2), and heart rate (371±12 vs. 277±19 beats/min), were each elevated (p&lt;0.01). In SHR, aging had no effect on these pressure parameters, or heart rate; whereas, in WKY, aging was associated with a significant increase in pulse pressure (31±1 mmHg; p=0.04) but no other pressure parameter. Mean (4.94±0.29 vs. 3.80±0.54 ml/min), maximum systolic (11.58±1.08 vs. 19.00±2.18), and minimum diastolic blood flow (0.62±0.73 vs. 1.90±0.34) were each reduced in young adult SHR vs. WKY (p&lt;0.05), while flow pulsatility was no different (3.23±0.73 vs. 3.43±0.44). Aging had no effect on these flow parameters in either SHR or WKY. Together, these data demonstrate that CCA blood flow is &gt;20% reduced in young adult SHR, and that this reduction is neither exacerbated nor attenuated at upper middle age. Given the important contribution of the internal branches of the CCAs to the total perfusion of the brain, these findings may be of pathophysiological and clinical relevance.Grant Funding Source: Supported by NSERC Canada

The Urotensin System Is Up-Regulated in the Pre-Hypertensive Spontaneously Hypertensive Rat

Urotensin II (UII) concentrations are raised both in humans with hypertension and in spontaneously hypertensive rats (SHR). Since the urotensin system acts to regulate glomerular filtration in the kidney it may play a greater role in the pre-hypertensive SHR in which renal dysfunction is known to precede the onset of severe hypertension. This study aimed to determine the renal actions and expression of the urotensin system in the young SHR. Intravenous rat UII (6 pmol. min-1. 100 g body weight-1) had no significant effect on GFR; however urotensin-related peptide (URP) reduced GFR (P<0.05) in 4-5 week old SHR. Administration of the UT antagonist SB-706375 evoked marked increases in GFR (baseline 0.38 ± 0.07 vs antagonist 0.76 ± 0.05 ml. min-1. 100 g body weight-1, P<0.05), urine flow and sodium excretion (baseline 2.5 ± 0.4 vs antagonist 9.1 ± 2.1 µmol. min-1. 100 g body weight-1, P<0.05) in the SHR. Normotensive Wistar-Kyoto rats showed little response to UT antagonism. Quantitative RT-PCR showed that neither UII nor UT mRNA expression differed between the kidneys of young SHR and WKY rats; however expression of URP was 4-fold higher in the SHR kidney. Renal transcriptional up-regulation indicates that URP is the major UT ligand in young SHR and WKY rats. Enhanced tonic UT activation may contribute to known renal dysfunction in pre-hypertensive SHR.

Comparison of 12-lipoxygenase expression in vascular smooth muscle cells from old normotensive Wistar-Kyoto rats with spontaneously hypertensive rats

Vascular aging and essential hypertension cause similar structural and molecular modifications in the vasculature. The 12-lipoxygenase (LO) pathway of arachidonic acid metabolism is linked to cell growth and the pathology of hypertension. Thus, elevated expression of 12-LO has been observed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). In the present study, we investigated the differences in 12-LO expression and activity between VSMCs from old normotensive Wistar-Kyoto rats (old WKY, 90-week old) and SHR (13-week old). The protein and mRNA expression of basal or angiotensin II (Ang II)-induced 12-LO in old WKY VSMCs were higher than those in SHR VSMCs. The degradation rate of 12-LO mRNA in old WKY VSMCs was slower than that in SHR VSMCs. However, basal or Ang II-induced 12-LO mRNAs in both old WKY and SHR VSMCs decayed more rapidly than that in young WKY (13-week old) VSMCs. Higher expression of 12-LO in old WKY VSMCs than in SHR VSMCs was correlated with the expression level of Ang II subtype 1 receptor (AT(1)R). The reduced levels of nitric oxide (NO) in old WKY and SHR VSMCs compared with young WKY VSMCs were similar, and there was no significant difference in NO production between old WKY and SHR VSMCs transfected with 12-LO siRNA. In addition, in contrast to the proliferation of SHR VSMCs, the proliferation of old WKY VSMCs was not dependent on 12-LO activation. These results suggest that the potential role of 12-LO in normotensive aging vasculature may be different from that in SHR vasculature.

Thrombospondin-1 in Early Flow-Related Remodeling of Mesenteric Arteries from Young Normotensive and Spontaneously Hypertensive Rats

We tested the hypotheses that TSP-1 participates in the initiation of remodeling of small muscular arteries in response to altered blood flow and that the N-terminal domain of TSP-1 (hepI) can reverse the pathological inward remodeling of resistance arteries from SHR.We measured (1) changes in gene/protein expression in MA of 6 week old WKY and SHR exposed to either increased (+ 100 %) or reduced blood flow (- 90 %) for 24-40 hours and (2) structural changes in MA of 12 week old SHR exposed for 3 days to hepI in organ culture.In both HF and LF of WKY, mRNA expression of eNOS, sGCα1 and PKG1β were significantly reduced (p < 0.05), whereas mRNA of TSP1 was markedly increased (p < 0.05). In MA of young SHR, similar results were obtained except that eNOS mRNA was not reduced in LF. Expression of TSP1 protein was significantly increased in LF of young WKY and SHR (p < 0.05). Exposure of MA of 12 week old SHR to hepI (1 µmol/L) resulted in a rapid lumen diameter increase (+ 12 ± 2% after 3 days) without alteration in vascular reactivity, distensibility, media surface area or cell number.These are the first observations of reduced gene expression of eNOS/sGC/PKG and increased expression of TSP1 at the initiation of arterial remodeling in young WKY and SHR, irrespective of its outward or inward outcome. Furthermore, a fragment of TSP-1 rapidly and directly reversed pathological inward arterial remodeling of SHR in vitro.

Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR

Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients; however, the precise mechanisms of its effects on hypertension are largely unknown. Therefore, this study aimed to investigate the mechanisms within the brain that can influence exercise-induced effects in an animal model of human essential hypertension. Young normotensive WKY rats and SHR were given moderate-intensity exercise for 16weeks. Blood pressure was measured bi-weekly by tail-cuff method. Animals were then euthanized; paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), important cardiovascular regulatory centers in the brain, were collected and analyzed by real-time RT-PCR, Western blot, EIA, and fluorescent microscopy. Exercise of 16-week duration attenuated systolic, diastolic, and mean arterial pressure in SHR. Sedentary SHR exhibited increased pro-inflammatory cytokines (PICs) and decreased anti-inflammatory IL-10 levels in the PVN and RVLM. Furthermore, SHR(sed) rats exhibited elevated levels of ACE, AT1R, and decreased levels of ACE2 and receptor Mas in the PVN and RVLM. Chronic exercise not only prevented the increase in PICs (TNF-α, IL-1β), ACE, and AT1R protein expression in the brain of SHR, but also dramatically upregulated IL-10, ACE2, and Mas receptor expression in SHR. In addition, these changes were associated with reduced plasma AngII levels, reduced neuronal activity, reduced NADPH-oxidase subunit gp91(phox) and inducible NO synthase in trained SHRs indicating reduced oxidative stress. These results suggest that chronic exercise not only attenuates PICs and the vasoconstrictor axis of the RAS but also improves the anti-inflammatory defense mechanisms and vasoprotective axis of the RAS in the brain, which, at least in part, explains the blood pressure-lowering effects of exercise in hypertension.

Magnetic resonance imaging quantification of regional cerebral blood flow and cerebrovascular reactivity to carbon dioxide in normotensive and hypertensive rats

Hypertension afflicts 25% of the general population and over 50% of the elderly. In the present work, arterial spin labeling MRI was used to non-invasively quantify regional cerebral blood flow (CBF), cerebrovascular resistance and CO(2) reactivity in spontaneously hypertensive rats (SHR) and in normotensive Wistar Kyoto rats (WKY), at two different ages (3 months and 10 months) and under the effects of two anesthetics, α-chloralose and 2% isoflurane (1.5 MAC). Repeated CBF measurements were highly consistent, differing by less than 10% and 18% within and across animals, respectively. Under α-chloralose, whole brain CBF at normocapnia did not differ between groups (young WKY: 61 ± 3ml/100g/min; adult WKY: 62 ± 4ml/100g/min; young SHR: 70 ± 9ml/100g/min; adult SHR: 69 ± 8ml/100g/min), indicating normal cerebral autoregulation in SHR. At hypercapnia, CBF values increased significantly, and a linear relationship between CBF and PaCO(2) levels was observed. In contrast, 2% isoflurane impaired cerebral autoregulation. Whole brain CBF in SHR was significantly higher than in WKY rats at normocapnia (young SHR: 139 ± 25ml/100g/min; adult SHR: 104 ± 23ml/100g/min; young WKY: 55± 9ml/100g/min; adult WKY: 71 ± 19ml/100g/min). CBF values increased significantly with increasing CO(2); however, there was a clear saturation of CBF at PaCO(2) levels greater than 70mmHg in both young and adult rats, regardless of absolute CBF values, suggesting that isoflurane interferes with the vasodilatory mechanisms of CO(2). This behavior was observed for both cortical and subcortical structures. Under either anesthetic, CO(2) reactivity values in adult SHR were decreased, confirming that hypertension, when combined with age, increases cerebrovascular resistance and reduces cerebrovascular compliance.

I030 Young spontaneously hypertensive rats (SHR) with chronic inhibition of nitric oxide exhibit similar aortic stiffness to old SHR

The old spontaneously hypertensive rat (SHR) is known to be a good experimental model that is highly analogous to essential hypertension in man but is costly and time consuming. Chronic inhibition of nitric oxide (NO) production in young adult SHR is an experimental model of malignant hypertension and chronic renal disease, very similar to old SHR1. Considering that acute or chronic decreases in NO production in normotensive rats increase arterial stiffness, we aimed to compare the aortic stiffness between SHR/L-NAME and old SHR. Male SHR (18 weeks old) received N-Nitro-L-Arginine Methyl Ester (L-NAME) 6 mg/kg/d in drinking water (SHR/L-NAME) for 2 weeks and were compared to SHR and Wistar Kyoto rats (WKY). Moreover, two groups are composed of 55 weeks aged SHR (Old SHR) and WKY (Old WKY). Under anesthesia, two catheters were introduced in the left common carotid artery and the left femoral artery to measure central and peripheral blood pressure and assess aortic stiffness with pulse wave velocity (PWV= distance between the tips of the two catheters / transit time), β-index (2.11×(PWV2/Diastolic blood pressure)), amplification and pulse pressure (PP). At 20 weeks, although SHR have higher central systolic blood pressure (SBP) than WKY (201±7 mmHg vs. 154±4 mmHg), no significant difference in PWV, PP, amplification and β-index was observed between these two groups. With age, old WKY have higher SBP (181±8 mmHg) and higher PWV (6.7±0.5 m/s) and β-index (0.70±0.08) than young WKY. Old SHR exhibit higher SBP (252±13 mmHg), PWV (9.3±0.7 m/s), PP (71±7 mmHg), β-index (1.08±0.16) than old WKY and young SHR (PWV=6.3±0.2 m/s, PP=39±3 mmHg and β-index=0.50±0.03). Young SHR, after 2 weeks of L-NAME administration, have higher SBP (238±10 mmHg) and aortic stiffness (PWV=8.6±0.6 m/s, PP=54±6 mmHg and β-index=0.89±0.14) than young SHR and possess the aortic stiffness observed in old SHR. In conclusion, our results indicate a close analogy in arterial stiffness between SHR/L-NAME and old SHR. Thus, this experimental model shows similarities to hypertensive humans and appears relevant to test new anti-hypertensive treatments.

Abstract 1627: Molecular Substrates of Carotid Body Chemoreceptor Activation

Carotid bodies are powerful regulators of sympathetic nerve activity in response to acidosis, hypoxia and hypercarbia. They are hypersensitive in heart failure and hypertension. The molecular determinants of their responsiveness and hypersensitivity are not fully understood. We recently reported that the acid sensitive channels ASICs and TASKs mediate early and late depolarizations, respectively, in isolated glomus cells ( Circ. Res. 2007 ; 101 : 1009 –19 ). In these studies we addressed two questions. The first is whether the enhanced carotid body hypersensitivity known to occur in spontaneously hypertensive rats (SHR) reflects enhanced activity of ASICs and TASKs. The results indicate that low pH ranging from 7.0 to 6.0 induced both an early rapid and a late sustained depolarization of glomus cells isolated from young (4 – 6 weeks) SHR and WKY rats, however the response was markedly enhanced in SHR. The values for rapid and sustained depolarization at pH 6.0 were 24.4 ± 3.7 and 39.2 ± 2.3 mV for SHR cells (n = 16), and 12.7 ± 2.3 and 29.5 ± 2.6 mV for WKY cells (n = 26), respectively. The ASIC blocker amiloride (200 μM) suppressed significantly the early depolarization to a level of 2.0 ± 1.2 mV (n = 7), but not the sustained depolarization which remained at 33.6 ± 3.6 mV (n = 7). mRNAs encoding ASIC1b and 3 and TASK1 and 3 were augmented by 150%, 75%, 100% and 20% respectively in carotid bodies of SHR compared to WKY. The second question is whether glomus cells are selectively responsive to low pH vs. oxygen deprivation with cyanide. In contrast to the response to low pH, that to cyanide (2 mM) was not enhanced in SHR (13.9 ± 2.5 mV; n = 7) compared to WKY (11.8 ± 3.3 mV; n = 7). Moreover in six WKY cells, three were as sensitive to cyanide (20.0 ± 3.9 mV) as they were to low pH (29.3 ± 4.6 mV) whereas three other cells that were very sensitive to low pH (29.2 ± 1.9 mV) had negligible responses to cyanide (4.2 ± 0.9 mV). We conclude: that in a genetic model of neurogenic hypertension (SHR), overexpression of acid sensitive ion channels (ASICs and TASKs) causes selective carotid body chemoreceptor hypersensitivity to low pH and may contribute to the development of hypertension; and that different glomus cell populations may be selectively sensitive to specific chemical stimuli (e.g. low pH vs. hypoxia).

Nɛ‐(Carboxymethyl)lysine during the Early Development of Hypertension

Advanced glycation end products (AGEs) are associated with hypertension. Whether N(epsilon)-(carboxymethyl)lysine (CML) contributes to the development of hypertension in young spontaneously hypertensive rats (SHR) remains to be established compared to WKY. We determined blood pressure, renal function, marker for oxidative stress (OS), and CML in young WKY rats and SHR. We found blood pressure was increased in SHR with no difference in renal function and OS compared to WKY. CML was elevated in plasma (2.3 +/- 0.3 vs. 1.3 +/- 0.2 micromol/L) and kidney (1.0 +/- 0.1 vs. 0.5 +/- 0.1 micromol/L) compared to WKY. Early CML accumulation may contribute to the development of hypertension potentially by inducing early renal inflammation independent of glomerular dysfunction or oxidative stress.

Elevated periarterial hydrogen peroxide in resistance vessels of spontaneously hypertensive rat (SHR) during rest and increased flow is reversed by apocynin

Flow‐mediated dilation is mediated by H2O2 in humans with arterial disease. We have shown that NO does not increase with elevated flow in resistance arteries of SHR and aged rats. These same vessels do not enlarge as collaterals. We tested the hypothesis that flow‐mediated H2O2 is upregulated in SHR, and reversed by the NADPH oxidase inhibitor, apocynin. Our mesenteric model was used in young WKY rats, SHR and SHR pre‐treated with apocynin (Apo‐SHR). H2O2‐specific electrodes were used to measure real‐time, in vivo mesenteric periarterial [H2O2]. Specificity of electrodes was verified with catalase. After H2O2 baseline measurement, collateral flow was increased 50–200 % by clamping adjacent arteries. Baseline H2O2 was significantly increased in SHR compared with WKY (26.2 ± 6.8 vs. 7.2 ± 0.9 μM, p&lt; 0.05) or Apo‐SHR (11.1 ± 3.2 μM). Successive increases in flow significantly increased periarterial [H2O2] in SHR from baseline (1 clamp, 46 ± 16 %, p&lt;0.05; 2 clamps, 61 ± 11 %, p&lt;0.05). Flow‐mediated [H2O2] increases were prevented in Apo‐SHR (−18 ± 5 %, 6 ± 23 %; 1 and 2 clamps respectively) and not observed in WKY. The results indicate that elevated baseline and flow‐mediated H2O2 production may originate from NADPH oxidase in SHR. Flow‐mediated H2O2 production may be a compensatory mechanism for vasodilation in SHR but may also be responsible for collateral growth impairment.Support: HL42898 and HL20605

Linked increased calcium and sodium in SHR of young age versus age-matched WKY

Linked increased calcium and sodium in SHR of young age versus age-matched WKY

Human-Antigen R (HuR) Expression in Hypertension

In aged spontaneously hypertensive rats (SHR), vasorelaxant responses to NO are attenuated compared with normotensive control rats (Wistar-Kyoto [WKY]) because of a decreased expression of the important NO receptor soluble guanylyl cyclase (sGC). Because the expression of sGC subunits alpha1 and beta1 is controlled at the post-transcriptional level by the mRNA-binding protein human-antigen R (HuR), we now assessed whether or not altered expression of HuR could account for downregulation of sGCalpha1 and sGCbeta1 in genetic hypertension. The expression of HuR (and sGCalpha1 and sGCbeta1) in aortas from aged SHR was significantly decreased at the mRNA and protein level compared with age-matched WKY rats, whereas expression of HuR was not different in prehypertensive young (2 months of age) SHR and age-matched WKY rats. The mRNA-binding activity of HuR in native aortic protein extracts from aged SHR was markedly reduced compared with normotensive WKY rats, as detected by RNA electrophoretic mobility shift analysis, using biotin-labeled adenine and uracil (AU)-rich element (ARE)-containing RNA probes from the 3'-untranslated region of sGCalpha1 and sGCbeta1. In contrast, ARE-binding activity was not different between prehypertensive young SHR and young WKY rats. In vitro RNA degradation assays using the same AU-rich sGC mRNA probes revealed an accelerated sGCalpha1 and sGCbeta1 mRNA decay in the presence of native protein extract from hypertensive SHR, which was less rapid with aortic protein from normotensive WKY rats. These findings suggest that in this animal model of genetic hypertension, the reduced expression of sGC subunits is mediated by downregulation of the sGC mRNA-stabilizing protein HuR.

Renovascular effects of sympathetic cotransmitters ATP and NPY are age-dependent in spontaneoulsy hypertensive rats

Hypertension is characterized by sympathetic overactivity. Neuropeptide Y (NPY) and ATP are cotransmitters of norepinephrine (NE) and regulate renovascular resistance. The present study analyzes sympathetic nonadrenergic neurotransmission in hypertensive (SH-SP) and normotensive (WKY) rats. In addition, adult and young hypertensive rats were compared to investigate the role of aging on sympathetic nonadrenergic cotransmission in hypertensive disease. Pressor responses to renal nerve stimulations (RNS) and drugs were measured on isolated perfused kidneys of young (8-10 weeks) and adult (18-24 weeks) WKY, and SH-SP rats. RNS evoked contractions at 1 Hz were resistant to blockade by the alpha-adrenoceptor antagonist phentolamine (1 microM) but abolished by the P2 receptor blocker suramin (100 microM). Compared to adult WKY, RNS-induced pressor responses were unchanged in adult SH-SP and young WKY, but significantly greater in young SH-SP rats. The NPY-Y1 receptor antagonist BIBP3226 (1 microM) reduced phentolamine-resistant pressor responses in adult and young WKY, young SH-SP, but not in adult SH-SP rats. In contrast to WKY and young SH-SP rats, exogenously perfused NPY (0.1 microM) was unable to potentiate RNS-induced, phentolamine-resistant pressor responses in adult SH-SP rats. NE and the stable ATP analogue alpha,beta-mATP increased the perfusion pressor response more potently in adult SH-SP than in WKY rats. Neuronally released NPY plays a major role in potentiating RNS-induced nonadrenergic pressor responses in kidneys of WKY and young SH-SP rats. In adult SH-SP rats NPY fails to enhance these responses. In this hypertensive model ageing seems to be associated with a loss of a modulatory role of renal NPY Y1 receptors. Since pressor responses to NE and ATP are higher in SH-SP animals, functional NPY-Y1 receptor downregulation might be an adaptive mechanism.

Age‐Related Analysis of Glucose Metabolism in Spontaneously Hypertensive and Normotensive Rats

This study was designed to investigate the effect of both hypertension and ageing on the efficiency of glucose metabolism. A 12-sample, 120 min intravenous glucose tolerance test (IVGTT) was applied to 36 rats: two groups of nine young (12 weeks) spontaneously hypertensive and Wistar Kyoto rats (Y-SHR and Y-WKY group, respectively) and two groups of nine old (40 weeks) SHR and WKY rats (O-SHR and O-WKY group, respectively). Insulinaemia and glycaemia data were interpreted in terms of estimates of glucose effectiveness, S(G), and insulin sensitivity, S(I), provided by the minimal model of glucose kinetics. The possible link between insulin resistance and hypertension was investigated by comparing Y-SHR vs. Y-WKY and O-SHR vs. O-WKY groups. Comparison of O-SHR vs. Y-SHR and O-WKY vs. Y-WKY groups enabled us to investigate the role of age in the development of abnormalities in glucose metabolism. No significant differences (P > 0.05) were observed in the mean S(G) and S(I) estimates between SHR and age-matched WKY groups. This finding indicates that exposure of SHR to high blood pressure levels does not necessarily lead to the development of insulin resistance and impaired glucose effectiveness. Similarly, no significant differences (P > 0.05) were observed in S(G) and S(I) estimates between old and young SHR and WKY groups. This finding indicates that, in this animal model of hypertension, insulin sensitivity and glucose effectiveness do not even deteriorate with ageing.

Renal Hemodynamics in Young and Old Spontaneously Hypertensive Rats during Intrarenal Infusion of Arginine Vasopressin

Background/Aims: To gain insight into the effect of arginine vasopressin (AVP) on renal hemodynamics in hypertensive rats, we investigated the vasoconstrictive response to AVP on total renal blood flow (RBF) and total and zonal glomerular filtration rate (GFR) in young and old spontaneously hypertensive rats (SHR). A hypothesis of increased AVP sensitivity in the juxtamedullary cortex of SHR was tested. Methods: Total RBF and total and zonal GFR were studied in 10- and 40-week-old SHR and normotensive Wistar-Kyoto rats (WKY). RBF was recorded by a flowmeter before infusion of AVP and immediately after injection of a bolus dose of 10 ng AVP. Whole kidney GFR and its intracortical distribution was measured by the tubular uptake of ¹²⁵I- and ¹³¹I-labelled aprotinin before and during a continuous infusion of AVP 5 ng/min. Ligand binding measurements of preglomerular V_1a receptors were performed in young and old rats. Results: RBF decreased by 43 ± 3% in 10-week SHR (9.2 ± 0.5 vs. 5.2 ± 0.3 ml·min^–1·g^–1), significantly more than 10-week WKY where RBF decreased by 35 ± 3% (9.6 ± 0.7 vs. 6.5 ± 0.5 ml·min^–1·g^–1) (p < 0.05). The effect of AVP on RBF was attenuated in 40-week-old rats where the decline in RBF was 29 ± 5% in SHR and 23 ± 4% in WKY (p > 0.05). GFR decreased by 6 ± 3% (1.03 ± 0.04 vs. 0.96 ± 0.04 ml·min^–1·g^–1, p < 0.05) in 10-week SHR and was unchanged in 10-week WKY (1.10 ± 0.07 vs. 1.08 ± 0.04 ml·min^–1·g^–1, p > 0.10). GFR decreased by 11 ± 10% in 40-week SHR and by 4 ± 4% in 40-week WKY (p > 0.05). AVP infusion significantly increased filtration fraction in all groups except 40-week SHR, indicating that AVP has the strongest vasoconstrictive effect on postglomerular vessels. The intrarenal distribution of GFR was unchanged in the normotensive and hypertensive groups. V_1a receptor density was upregulated in young SHR compared to young WKY (p < 0.05), but downregulated in old compared to young SHR (p = 0.05). Conclusion: The results indicate that AVP sensitivity is not increased in the juxtamedullary cortex in SHR and the reduced vasoconstrictive effect in old SHR is due to a reduced density of V_1a receptors.

Aging and Chronic Hypertension Decrease Expression of Rat Aortic Soluble Guanylyl Cyclase

We analyzed the influence of aging and genetic hypertension on the function and expression of soluble guanylyl cyclase (sGC) in the aortas of prehypertensive and old spontaneously hypertensive rats (SHR) as well as in age-matched normotensive Wistar-Kyoto rats (WKY). The expression of heterodimeric sGC (alpha(1) and beta(1)) was assessed at the mRNA and protein level, and its function was assessed by the relaxant responses of phenylephrine-contracted endothelium-denuded aortic rings to the nitric oxide (NO) donor sodium nitroprusside. The vasodilator potency of sodium nitroprusside was significantly reduced (P<0.05) with age (3- to 6-fold increase in the EC(50) in old WKY and SHR compared with their young counterparts) as well as with hypertension (3-fold increase in old SHR compared with age-matched WKY), whereas the vasodilator potency of sodium nitroprusside did not differ between young SHR and WKY. A similar influence of aging and hypertension on NO-stimulated GC activity was revealed at the GC expression level: Whereas the beta(1) protein content was similar in young rats of both strains, old WKY exhibited 60% lower and old SHR exhibited 80% lower beta(1) subunit protein compared with young rats (P<0.05). Moreover, the abundance of alpha(1) and beta(1) mRNA (assessed by reverse transcriptase-polymerase chain reaction) was similar in young rats but was 2.5-fold (alpha(1)) and 4.3-fold (beta(1)) lower in old SHR compared with old WKY. In conclusion, our findings show that both aging and hypertension decrease sGC expression and its NO-dependent activation in aortic tissue. Downregulation of sGC may therefore contribute to arterial dysfunction in senescence and chronic hypertension.