Young SHR Research Articles

The antihypertensive and antihypertrophic effect of lycopene is not affected by and is independent of age

• Hypertension and aging in SHR induces cardiac, vascular and renal alterations. • Lycopene decreased blood pressure in young and adult SHR. • Lycopene reduced hypertrophy and fibrosis in heart, kidney and aorta. • Lycopene had beneficial effect on oxidative stress on SHR. Hypertension of SHR appears at an early age and progressively increases. This study aimed to evaluate the effect of lycopene in SHR with mild hypertension (young rats) or with high level of hypertension (adult rats). Four-weeks treatment with 10 mg/kg/day of lycopene progressively decreased blood pressure in young (from 144 ± 2 to 119 ± 3 mmHg) and adult (from 177 ± 5 to 159 ± 7 mmHg) SHR. Heart and renal hypertrophy and fibrosis were increased in adult compared to young SHR and lycopene, regardless of the age of the rats, improved the injury in both organs. Although aging induced morphological and functional alterations in the aorta, the treatment was only effective in preventing the former. Lycopene helped to improve all the parameters linked to oxidative stress determined in this study. In conclusion, lycopene treatment improved the age-associated harmful changes in hypertension, cardiovascular and renal remodelling, and indicators of oxidant-antioxidant systems in both young and adult SHR.

Abstract 15555: Potential Cure for Hypertension? The Effect of Crispr Genome Editing

Hypertension is recognized as one of the largest contributors to the global burden of disease. Unfortunately, for the majority of patients, hypertension is poorly controlled due, in part, to poor medical compliance resulting from the need to take one or more drugs, often several times daily. An ideal therapy would be administered a single time but yield long-term blood pressure control. Under the appropriate condition, we hypothesize that this modality could potentially lead to a cure for hypertension. In this study, we investigated whether that sustained, and possibly life-long, reductions in blood pressure could be achieved via Crispr-Cas9 mediated disruption of a key gene in the RAAS, Angiotensinogen (AGT). In vitro, we demonstrated that expression of Crispr-Cas9 system in the BRL 3A rat liver cell line led to a significant reduction in AGT protein levels (75% reduction, N=3, P<0.01). Delivery of the Crispr-Cas9 system into the liver via systemic delivery of the hepatocyte-targeting AAV8 reduced both AGT mRNA (70% reduction, N=5, P<0.01) and circulating AGT protein (50% reduction, N=5, P<0.01) and Ang II levels (33% reduction, n=5, P<0.01). In the SHR model of hypertension, Crispr-Cas9 mediated loss of AGT expression reduced systolic blood pressure in adult animals with established hypertension (30-35mmHg decrease in both females and males, N=12, P<0.001). Importantly, AGT gene disruption also prevented the spontaneous development of hypertension in young pre-hypertensive SHR (SHR control: 55-60mmHg increase in systolic blood pressure both female and male SHR; SHR plus Crispr-Cas9: 30-33 mmHg increase. N=12. P<0.001). Importantly, in all the models studied, reductions in blood pressure were sustained for at least 4 months. Importantly, the partial disruption of the hepatic AGT gene was sufficient to control hypertension but did not block the renin-angiotensin response to cardiovascular stress such as sodium depletion. In summary, we have demonstrated that targeting the Crispr-Cas9 system to RAAS reduces blood pressure and may be a potential therapy to achieve safe and sustained, possibly life-long, control of hypertension and this approach maybe a cure for hypertension.

Nicotine Mediates CD161a+ Renal Macrophage Infiltration and Premature Hypertension in the Spontaneously Hypertensive Rat.

Renal inflammation contributes to the pathophysiology of hypertension. CD161a+ immune cells are dominant in the (SHR) spontaneously hypertensive rat and expand in response to nicotinic cholinergic activation. We aimed to phenotype CD161a+ immune cells in prehypertensive SHR after cholinergic activation with nicotine and determine if these cells are involved in renal inflammation and the development of hypertension. Studies used young SHR and WKY (Wistar-Kyoto) rats. Splenocytes and bone marrow cells were exposed to nicotine ex vivo, and nicotine was infused in vivo. Blood pressures, kidney, serum, and urine were obtained. Flow cytometry, Luminex/ELISA, immunohistochemistry, confocal microscopy, and Western blot were used. Nicotinic cholinergic activation induced proliferation of CD161a+/CD68+ macrophages in SHR-derived splenocytes, their renal infiltration, and premature hypertension in SHR. These changes were associated with increased renal expression of MCP-1 (monocyte chemoattractant protein-1) and VLA-4 (very-late antigen-4). LLT1 (lectin-like transcript 1), the ligand for CD161a, was overexpressed in SHR kidney, whereas vascular cellular and intracellular adhesion molecules were similar to those in WKY. Inflammatory cytokines were elevated in SHR kidney and urine after nicotine infusion. Nicotine-mediated renal macrophage infiltration/inflammation was enhanced in denervated kidneys, not explained by angiotensin II levels or expression of angiotensin type-1/2 receptors. Moreover, expression of the anti-inflammatory α7-nAChR (α7-nicotinic acetylcholine receptor) was similar in young SHR and WKY rats. A novel, inherited nicotinic cholinergic inflammatory effect exists in young SHR, measured by expansion of CD161a+/CD68+ macrophages. This leads to renal inflammation and premature hypertension, which may be partially explained by increased renal expression of LLT-1, MCP-1, and VLA-4.

Abstract P132: Role of NO/NOS System in Blood Pressure Regulation in Postmenopausal Female SHR with Hormone Replacement Therapy

Estradiol (E 2 ) and testosterone (T) stimulate synthesis of nitric oxide (NO). Since the WHI study, postmenopausal women are given E 2 and/or T supplements to alleviate the symptoms of menopause. NO is a key regulator of blood pressure (BP), mediating endothelial and vascular function. Studies from our lab showed that E 2 plays little role in BP control in young female SHR; however, whether E 2 replacement with or without T would reduce BP in postmenopausal female SHR is not known. If E 2 does not reduce BP in old female SHR, it is possible that there is a defect in the NO system that prevents the vasodilatory E 2 effect mediated by NO. The hypothesis tested in this study was that E 2 /T replacement would reduce BP in old female SHR, and if not, the mechanism responsible is a deficient NO system that is incapable of upregulating in response to E 2 . After baseline (B) mean arterial pressure (MAP; telemetry), female SHR (19 mos, n=5) were implanted with 17-β E 2 (0.1 mg/pellet) and T (5mm in silastic tubes) and MAP was measured. After a transient reduction in MAP over 2-3 days (B: 175±5; E 2 +T: 161±4 mm Hg, p<0.05), MAP returned to baseline levels by day 4 (176±5 mm Hg). These data suggest that the acute vasodilatory response to E 2 /T in old female SHR was intact. On day 8 T tubes were removed, and MAP was measured for additional 16 days. Removal of T had no effect on BP (175±5 mm Hg). To evaluate NO system, rats were given: 1) 2% L-Arginine (L-Arg, 21 d); 2) 0.1% sodium nitrite (NaNO 2 , 6 d); 3) nitro-L-arginine methyl ester (L-NAME, 4mg/kg/d, 5 d). L-Arg supplement failed to change MAP (B: 175±5, L-Arg: 176±5 mm Hg, p<NS). In contrast, NaNO 2 did decrease MAP (B: 176±5, NaNO 2 : 161±3 mm Hg, p<0.05), suggesting a deficient endogenous synthesis of NO but the ability of the old female SHR to respond to an NO donor. L-NAME increased MAP (B: 176±5; L-NAME: 189±3 mm Hg, p<0.05). In total, these data suggest that the NO system in old female SHR is capable of responding appropriately to NO donors or complete blockade. However, the lack of response to L-Arg suggests a deficiency in the ability to normally synthesize NO, and thus may in part be responsible for the lack of a depressor response to E 2 , and therefore, may contribute to the elevated BP in old female SHR. Supported by NIH-R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM).

Abstract P133: Melanocortin 4 Receptor is Required for the Effect of Testosterone Supplement on Metabolic Parameters and Blood Pressure

Melanocortin 4 receptor (MC4R) activation causes appetite suppression and sympathetic nervous system activation. Blockade of MC4R increases food intake and in some cases, decreases BP. MC4R has been implicated as playing a role in obesity and hypertension. Previously we observed that testosterone supplementation in male Zucker rats improved insulin resistance and the characteristics of metabolic syndrome, but increased BP. Similarly, testosterone increases BP in male SHR, and blockade of the MC4R reduces BP in both young and old male SHR. In the present study we tested the hypothesis that testosterone supplementation (T) requires activated MC4R to attenuate metabolic parameters, and to increase BP. Male obese MC4R -/- and Wistar Hannover rats (WT) were treated with T (MC4R-KO+T vs WT+T: 22 mg/10 mm silastic pellet) or placebo (empty pellets; MC4R-KO+P, and WT+P), beginning at 10 wks of age for 6 weeks. In WT animals, T reduced body weight by 13% compared to placebo (367 gr vs 432gr; p<0.05), but had no effect on food intake (WT+P: 19±7 gr/d vs WT+T: 16±5 gr/d, n=3, p:NS). In MC4R-KO rats, body weight and food intake were similar in both placebo and T rats (BW: 555±20 gr vs 526±18 gr, and FI: 26±1 gr vs 28±2; n=3, p:NS, respectively). T reduced fat mass (measured by ECHO-MRI ® ) in WT+T by 38% compared to placebo (50±2.9 gr vs 31±3.2 gr; p<0.05); however, there was no effect on fat mass in MC4R-KO + T (149±8 gr vs 147±11 gr; n=3, p:NS). Lean mass (by ECHO-MRI ® ) was not affected by T in either MC4R-KO+T or WT+T (p:NS). Finally, unlike our previous studies in obese Zucker and SHR males, T failed to affect mean arterial pressure or heart rate (by telemetry) in MC4R-KO+T rats compared to control MC4R-KO+P (MAP: 122±2 mmHg vs 122±1 mmHg; n=3, p:NS; HR:309±5 bpm vs 320±17 bpm; n=3, p:NS). These data suggest that the effect of T on metabolic parameters and BP may be mediated at least in part by MC4R since in the absence of active MC4R, T is not able to affect either body composition or BP. Also, these data gave a potential new insight into the mechanism by which T contributes to BP control and adipose tissue regulation. More studies are necessary to clarify the role of MC4R in mediating the effects of testosterone. Supported by NIH-R01HL66072, R01HL69194, PO1HL51971 (JFR), 14POST18640015 (ROM).

Pial Collateral Reactivity During Hypertension and Aging: Understanding the Function of Collaterals for Stroke Therapy.

We investigated vasoactive properties of leptomeningeal arterioles (LMAs) under normotensive conditions and during hypertension and aging that are known to have poor collateral flow and little salvageable tissue. LMAs, identified as distal anastomotic arterioles connecting middle and anterior cerebral arteries, were studied isolated and pressurized from young (18 weeks) or aged (48 weeks) normotensive Wistar Kyoto (WKY18, n=14; WKY48, n=6) rats and spontaneously hypertensive rats (SHR18, n=16; SHR48, n=6). Myogenic tone and vasoactive responses to pressure as well as endothelial function and ion channel activity were measured. LMAs from WKY18 had little myogenic tone at 40 mm Hg (8±3%) that increased in aged WKY48 (30±6%). However, LMAs from both WKY groups dilated to increased pressure and demonstrated little myogenic reactivity, a response that would be conducive to collateral flow. In contrast, LMAs from both SHR18 and SHR48 displayed considerable myogenic tone (56±8% and 43±7%; P<0.01 versus WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial nitric oxide and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. This study shows for the first time that LMAs from young and aged SHR are vasoconstricted and have impaired vasodilatory responses that may contribute to greater perfusion deficit and little penumbral tissue. These results also suggest that therapeutic opening of pial collaterals is possible during middle cerebral artery occlusion to create penumbral tissue and prevent infarct expansion.

Osteogenic markers are reduced in bone-marrow mesenchymal cells and femoral bone of young spontaneously hypertensive rats

AimsSpontaneously hypertensive rats (SHR) and normotensive rats (W) has significant changes in bone metabolism. The purpose of this study was to investigate whether, the genetic predisposition, is sufficient to induce changes in the osteoblast differentiation and osteogenic markers in the BMSCs or in the femoral bone. For this we use young SHR rats without hypertension, but, with genetic predisposition in compared with young W. Main methodsBMSCs were cultured in a proliferation medium (MEM) or osteogenic medium. Osteogenic differentiation was analyzed by proliferation, total protein, alkaline phosphatase, mineralization, and the mRNA expression of RUNX-2, β-cathenin, osterix, bone morphogenetic protein-2(BMP-2), osteocalcin (OCN), bone sialoprotein (BSP), collagen type I (Col I), and osteopontin (OPN). Key findingsOsteoblast differentiation in SHR BMSCs (SHRC) had an increased proliferation compared with W BMSCs (WC). After osteogenic induction, there was greater reduction in proliferation in SHR (SHROM) than in W, in the same condition (WOM). On day 7, although no significant difference in the ALP activity was observed between SHROM and WOM, poor mineralization and osteoblast differentiation was noted in SHROM. The Osterix and β-catenin are involved in the reduced osteoblast differentiation in SHROM. The decreased expression of osteoblast-associated proteins such as OCN, BSP, COL I and OPN revealed poor quality of extracellular matrix (ECM) in SHROM. In the femoral bone, the immunostaining of COL1, BALP, OPN and OCN in SHR was decreased compared with the W. TRAP-positive immunoreactions were observed in major extension in the SHR femur. SignificanceThis study is the first to compare osteoblast differentiation in vitro and femoral bone from SHR and W rats. Our results demonstrated that young SHR (4weeks old), without hypertension, but with genetic predisposition, had alterations in osteoblast differentiation of BMSCs and in the femoral bone when compared with their progenitor strain, W.

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.

Abstract P081: Tempol Decreases Blood Pressure in Aged Female SHR When Treated With Acetazolamide

Oxidative stress contributes to the development and maintenance of hypertension in male rodents, but studies in females have rarely shown a reduction in blood pressure (BP) with antioxidants. Tempol, a superoxide dismutase mimetic, decreases BP in young male SHR, but fails to reduce BP in either young or old female SHR, despite the fact that females have similar or higher levels of oxidative stress markers. The reason for the sex difference in the response to tempol remains unclear. Acetazolamide inhibits carbonic anhydrase in the proximal tubule thus increasing sodium delivery to the distal nephron, and thereby should increase distal oxidative stress. Acetazolamide was used to test the hypothesis that with increased sodium delivery to the distal nephron, tempol would reduce the BP in aging female SHR. Female SHR, 20-22 mos old, were divided into three groups (n=4-6/grp): Control (C), Acetazolamide (A), and Acetazolamide+Tempol (A+T). After baseline mean arterial pressure (MAP; telemetry), rats received vehicle (C) or acetazolamide (A and A+T). On day 8, rats in C and A+T groups were given tempol (30 mg/kg) for 11 days. Baseline MAP was similar (C: 170±7; A: 182±4; A+T: 172±6 mm Hg, p=NS). Tempol had no effect on MAP in C+T, but reduced MAP in A+T group (C+T: 169±1; A: 171±1; A+T: 151±5 mm Hg; p&lt;0.005 A+T vs A, C+T). Basal renal oxidative stress measured by lucigenin chemiluminescence was not different in the groups; NADPH-stimulated oxidative stress was decreased in A+T compared to A and C+T (C+T: 641.8±72.2; A: 499.3±18.3; A+T: 406.2±56.3 RLU/mg/min; p&lt;0.05, A+T vs A, C+T). Plasma total antioxidant capacity was increased by tempol only in A+T rats (C+T: 59.07±9.67; A: 69.01±4.66; A+T: 118.24±18.38 nmol/μl; p&lt;0.05, A+T vs A, C+T). Thus tempol is capable of modestly reducing MAP in aging female SHR when proximal sodium reabsorption is blocked. The data suggest that oxidative stress-mediated BP control is dependent on increased sodium delivery to the distal nephron. Because hypertension in male SHR is attenuated with tempol alone, but not in females, taken together, the data suggest sex differences in sodium handling and thus localization of oxidative stress production in the kidneys of SHR. Supported by NIH-R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM).

Abstract P039: Testosterone Increases BP in Male SHR by Activating the Renin-angiotensin System: A Cautionary Tale for “Low T” Supplements

Testosterone supplements are widely prescribed for men in the US to improve overall quality of life by increasing libido and sexual performance and protecting against osteoporosis. Whether testosterone supplements affect CVD either positively or negatively is not clear since study results are controversial. Previously, we showed that chronic testosterone supplements cause hypertension in obese male Zucker rats but improves inflammation and metabolic syndrome symptoms. We also found that testosterone supplements in young male SHR further increase their BP, and that castration attenuates the hypertension. In the present study we followed up on these previous studies and tested the hypothesis that, because the SHR are lean, chronic testosterone supplements will increase lean body mass but increase the BP via a renin angiotensin system (RAS) mechanism. Male SHR were treated with testosterone (8 mg/10 mm silastic pellet implanted sc) or placebo (empty pellets sc), beginning at 12 weeks of age for 8 weeks. Fat mass and lean mass were measured by ECHO MRI. Contrary to our hypothesis, testosterone supplements had no effect on lean mass, but reduced fat mass by 49% (4.9±0.6 vs 2.5±0.4 %BW, p&lt;0.05). Baseline mean arterial pressure (MAP by telemetry) was higher in testosterone supplemented rats than placebo controls (157±0.4 vs. 147±0.7 mmHg, p&lt;0.05). Enalapril (200 mg/L) reduced MAP by 25% in testosterone group (157±0.4 vs 118±6 mmHg, p&lt;0.05) and by 14% placebo rats (148±0.5 vs 127±5 mmHg, p&lt;0.05). MAPs after enalapril were similar between placebo and testosterone-treated rats. These data suggest that the RAS contributes to the elevated BP in testosterone supplemented male SHR. The data also suggest that caution should be taken in prescribing hypertensive men with testosterone supplements. Supported by NIH-R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM), EFF Endocrine Res Grant (LLY).

Abstract 352: Evaluation of Structural, Mechanical and Functional Properties in Small Arteries of Spontaneously Hypertensive Rats Before and After the Development of High Blood Pressure.

Structural, mechanical and functional adjustments occur in small mesenteric arteries (SMA) of hypertensive models. However, the role of these properties to trigger hypertension is unknown. As expected, the systolic blood pressure was higher in adult (A, 6-month old) male SHR as compared to Wistar-Kyoto rats (WKY) (WKYA: 125±1.1 vs SHRA: 187±3.3 mmHg*); however, it was similar in young (Y, 6-week old) SHR as compared to age-matched WKY (WKYY: 117±1.8 vs SHRY: 120±2.1 mmHg). The 3rd order mesenteric arteries were mounted in a pressure myograph to analyze the structural [lumen diameter (L), cross sectional area (CSA), wall/lumen ratio (W/L)] and mechanical properties [β, representing wall stiffness]. Endothelium-dependent relaxation to acetylcholine (ACh, 10-10-10-5 M) or -independent relaxation to sodium nitroprusside (SNP, 10-9-10-4 M) were evaluated in SMA using wire myography. At the passive condition (Ca2+-free solution) and intraluminal pressure of 160 mmHg, the L was lower in SMA of both SHR (WKYY: 294±12.0 vs SHRY: 241±4.3*; WKYA: 353±4.7 vs SHRA: 283±6.2 μm*); while the W/L ratio was higher in SHR as compared to age-matched WKY. CSA was similar between age-matched groups. β value was higher in SHR independently of age (WKYY: 5.8±0.4 vs. SHRY: 7.8±0.4*; WKYA: 4.7±0.1 vs SHRA: 6.7±0.4*). The collagen area evaluated by picrosirius red staining was higher in SMA of SHRA as compared to WKYA (WKYA: 15±2.4 vs SHRA: 26±1.8%*), but it did not change in young rats. ACh-induced maximal relaxation was similar in SMA from young groups (WKYY: 93±3.8 vs SHRY: 92±3.1%); however, in SHRA ACh elicited a biphasic curve inducing contraction at concentrations higher than 10-7M, which was not observed in WKYA. Relaxation to SNP did not change among groups. Reactive oxygen species analyzed by dihydroethidium was higher in SMA of SHRA as compared to WKYA (WKYA: 100±3.7 vs SHRA: 126±10.3% of integrated density*), but did not change in young SMA. Although SMA of SHRY present eutrophic inward remodeling and wall stiffening, it does not present collagen deposition, oxidative stress or endothelial dysfunction as observed in SHRA; suggesting that vascular remodeling and wall stiffness of SMA are not sufficient to trigger hypertension, at least when endothelial function is preserved.

Altered protein phosphatase 1 (PP1) activity and expression contribute to the differential regulation of proximal tubule NHE3 before and after onset of hypertension in SHR

We have previously demonstrated that NHE3 activity is higher in the proximal tubule of young pre‐hypertensive SHR (Y‐SHR) compared to adult hypertensive SHR (A‐SHR). This fall in NHE3 activity with blood pressure increase was associated with a higher ratio of phosphorylated NHE3 at serine 552 to total NHE3 (P‐NHE3/ total). The present study was undertaken to test the hypothesis that the increased levels of phosphorylated NHE3 at the PKA consensus site serine 552 was due to an increase of PKA activity, or a decrease of PP1 activity, or both. To confirm that PKA‐mediated phosphorylation of NHE3 regulation is altered in SHR, we examined the effect of 6MB‐cAMP (an cAMP analog that specifically activates PKA) on both phosphorylation levels and activity of NHE3 in young and adult SHR. We found that the 6MB‐cAMP‐induced increase in the levels of NHE3 phosphorylation at the serine 552 was much higher in the renal cortex from Y‐SHR than from A‐SHR (179 ± 14 vs. 36 ± 4%, P &lt; 0.001). Likewise, the inhibitory effect of 6MB‐cAMP on NHE3‐mediated bicarbonate reabsorption was much more pronounced in proximal tubule from Y‐SHR than from A‐SHR (65 ± 10 vs. 23 ± 9%, P &lt; 0.01). PKA activity remained unchanged between Y‐SHR and A‐SHR. On the other hand, Y‐SHR exhibited higher PP1 activity than A‐SHR (1640 ± 107 vs. 940 ± 119 pM/μg, P &lt; 0.01). Furthermore, PP1α expression was decreased in the renal cortex of A‐SHR compared to Y‐SHR (32 ± 8 %, P &lt; 0.01). We conclude that the differential regulation of proximal tubule NHE3 activity and phosphorylation levels before and after onset of hypertension in SHR is, at least in part, due to changes in the activity and expression of PP1.Supported by FAPESP

Effects of PPAR γ Agonist Pioglitazone on Redox-Sensitive Cellular Signaling in Young Spontaneously Hypertensive Rats.

PPARγ receptor plays an important role in oxidative stress response. Its agonists can influence vascular contractility in experimental hypertension. Our study was focused on the effects of a PPARγ agonist pioglitazone (PIO) on blood pressure regulation, vasoactivity of vessels, and redox-sensitive signaling at the central (brainstem, BS) and peripheral (left ventricle, LV) levels in young prehypertensive rats. 5-week-old SHR were treated either with PIO (10 mg/kg/day, 2 weeks) or with saline using gastric gavage. Administration of PIO significantly slowed down blood pressure increase and improved lipid profile and aortic relaxation after insulin stimulation. A significant increase in PPARγ expression was found only in BS, not in LV. PIO treatment did not influence NOS changes, but had tissue-dependent effect on SOD regulation and increased SOD activity, observed in LV. The treatment with PIO differentially affected also the levels of other intracellular signaling components: Akt kinase increased in the the BS, while β-catenin level was down-regulated in the BS and up-regulated in the LV. We found that the lowering of blood pressure in young SHR can be connected with insulin sensitivity of vessels and that β-catenin and SOD levels are important agents mediating PIO effects in the BS and LV.

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.

Inhibition of dipeptidyl peptidase IV by sitagliptin increases GLUT4 expression in insulin‐sensitive tissues of SHR

The goal of the present study was to investigate whether the dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin, which has been shown to exert both anti‐hyperglycemic and anti‐hypertensive actions, modulates GLUT4 expression in skeletal and cardiac muscles of SHR. To this end, male SHR at 5 and 20 weeks of age were treated with either sitagliptin (40 mg/kg twice daily) or vehicle for the period of ten days. Age‐matched Wistar Kyoto (WKY) rats were used as normotensive controls. Sitagliptin treatment inhibited the activity of DPPIV in the plasma of both young and adult SHR and also decreased the circulating levels of the enzyme. Sitagliptin only suppressed blood pressure rising in Y‐SHR. Levels of plasma insulin were not significantly different between the strains at young age. In contrast, adult SHR displayed enhanced plasma insulin concentration with fasting normoglycemia. Inhibition of DPPIV by sitagliptin restored plasma insulin levels of A‐SHR. GLUT4 in the plasma membrane (PM) was lower in Y‐SHR than in Y‐WKY in the heart, the slow‐twich oxidative soleus, the fast‐twich glycolytic EDL and the fast‐twich glycolytic‐oxidative gastrocnemius. DPPIV inhibition normalized PM GLUT4 in myocytes from Y‐SHR. Reduction of GLUT4 PM expression in the skeletal and cardiac myocytes was more pronounced in A‐SHR vs. A‐WKY than in Y‐SHR vs. YWKY. Inhibition of DPPIV by sitagliptin increased GLUT4 PM, total protein and GLUT4‐mRNA expression in the heart, soleus, EDL and gastrocnemius of treated‐A‐SHR to levels higher than those found in the A‐WKY. Collectively, our data demonstrate that DPPIV inhibition upregulates GLUT4 in skeletal and cardiac muscles of SHR.

The role of the renal sympathetic nerves in a model of postmenopausal hypertension

The mechanisms responsible for the increase in blood pressure (BP) in postmenopausal women are unknown. Our preliminary studies suggest that the sympathetic nervous system may contribute to the hypertension (HT) in aging female SHR. The aim of this study was to determine whether the renal nerves contribute to HT in old and young female SHR. Female SHR (18 and 3 mos of age, respectively, n=4/grp) underwent uninephrectomy, and two weeks later, underwent unilateral renal denervation (O‐RD and Y‐RD) or sham surgery (O‐S and Y‐S) and telemetry transmitter implantation. After two weeks recovery, mean arterial pressure (MAP) and heart rate (HR) were recorded for 5 days. MAP was higher in O‐S than other groups (O‐S:167.5±2.4 vs O‐RD:138±2.7 mmHg; p&lt;0.001; and Y‐S:140.6±6.3 vs Y‐RD:122.1±3.9 mmHg, p&lt;0.001). The reduction in MAP after denervation was greater in O‐RD (O‐RD:29.5±0.5 mmHg, vs Y‐RD:17.0±0.9 mmHg, p&lt;0.001). Renal NE content was significantly decreased compared to shams (O‐S: 201.1±30.3 pg/mg vs O‐RD: 35.6±10.3 pg/mg wet weight, p&lt;0.001; and Y‐S:239±24.5 pg/mg vs Y‐RD:18.1±11.4 pg/mg wet weight, p&lt;0.001). These data indicate that the renal nerves play a key role in the control of BP in pre‐ and post‐cycling SHR. The data also suggest that the renal nerves contribute to HT more in old females than young.

Induction of albuminuria and kidney damage in SHR by transfer of chromosome 8 from Munich Wistar Frömter rats

Inbred Munich Wistar Frömter [MWF/FubRkb (RGD:724569), MWF] rats develop progressive albuminuria with age that is under polygenetic influence. We previously identified a major albuminuria quantitative trait locus (QTL) on rat chromosome (RNO)8 in MWF. To test the independent role of QTL(s) for albuminuria development on RNO8, we generated a consomic SHR-Chr 8(MWF)/Rkb (SHR-8(MWF)) strain by transferring RNO8 from MWF into the albuminuria-resistant background of the spontaneously hypertensive rat [SHR/FubRkb (RGD:631696; SHR)]. Young male MWF, SHR, and SHR-8(MWF) were sham-operated or unilaterally nephrectomized (Nx) at 6 wk and followed up to 24 wk of age, respectively. Systolic blood pressure was significantly lower in SHR-8(MWF) Sham compared with SHR Sham (-19.4 mmHg, P = 0.03) at 24 wk. In contrast, transfer of MWF-RNO8 into SHR induced a significant elevation of urinary albumin excretion (UAE) between weeks 12 and 24 in SHR-8(MWF) compared with SHR Sham animals (P < 0.0001, respectively). Nx resulted in a significant increase in UAE in both strains during follow-up (P < 0.0001, respectively), with significant higher values in SHR-8(MWF) compared with SHR (P < 0.005, respectively). Renal structural changes as determined by glomerulosclerosis (GSI) and tubulointerstitial damage index (TDI) were significantly higher in consomic animals either at Sham (TDI) or Nx (GSI) conditions (P < 0.05, respectively). These data confirm the independent role of MWF QTL(s) on RNO8 for both albuminuria and structural kidney damage. Moreover, this study shows for the first time the induction of albuminuria by transferring one or more albuminuria QTL into a resistant recipient background in a consomic rat strain.

N-003 HIGH L-CITRULLINE TO L-ARGININE RATIO RELATES TO BLOOD PRESSURE ABNORMALITIS IN CHILDREN WITH CHRONIC KIDNEY DISEASE

Background Cardiovascular disease is a major cause of death in children with chronic kidney disease (CKD). The 24-hr ambulatory blood pressure monitoring (ABPM) is a superior predictor of cardiovascular outcome when compared to clinic BP measurement. In the kidney, L-citrulline (Cit) can be converted to L-arginine (Arg) to generate NO. We found that restoration of renal Arg availability is associated with lowering BP in young SHR. Thus we hypothesize that plasma Cit-to-Arg ratio reflects renal Arg availability, which relates to BP abnormalities in children with early chronic kidney disease (CKD). Methods The CKD staging is defined by the K/DOQI guideline. The eGFR was calculated by the Schwartz formula on the basis of height and serum creatinine. Plasma Arg and Cit levels were measured by HPLC. Subjects wore a 24-hr ABPM. Results We totally enrolled 22 children with stages 1–2 CKD. Approximately 80% (18/22) of children with early CKD had BP abnormalities. We found plasma levels of Cit and Arg, and Cit-to-Arg ratio were not different between children with CKD 1 (n = 8) and those with CKD 2 (n = 14). However, plasma Cit level and Cit-to-Arg ratio both were significantly higher in patients with abnormal ABPM profile than those with normal profile, including BP load and nocturnal BP-dipping. In addition, we found that plasma Cit level showed a significantly positive correlation with nighttime systolic (r = 0.464, P = 0.029) and diastolic BP (r = 0.479, P = 0.024), and diastolic BP load (r = 0.608, P = 0.003). Similarly, plasma Cit-to-Arg ratio was positively correlated with nighttime systolic (r = 0.52, P = 0.013) and diastolic BP (r = 0.564, P = 0.006), and diastolic BP load (r = 0.704, P < 0.001). Conclusions In conclusion, high Cit level and Cit-to-Arg ratio in the plasma are associated with BP abnormalitis on ABPM in children with CKD. Plasma Cit-to-Arg ratio may serve as a marker of cardiovascular outcome in children and adolescents with early CKD.

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.

Aging‐Related Decrease in Bax is Associated with Reduced Susceptibility to ACE Inhibitor‐Induced Remodeling in the Aorta of Spontaneously Hypertensive Rats

A 2‐week treatment with angiotensin converting enzyme inhibitor (ACEI) in young adult SHR reverses hyperplasia via apoptosis of aortic smooth muscle cells. The present study examined whether the same effect is observed in older rats. Male SHR (young: 11wks, mature: 26wks) were treated with either tap water or enalapril. Mature rats showed a significant increase in aortic cross‐sectional area, media thickness, lumen diameter, and media to lumen ratio but no change in collagen deposition. Enalapril significantly reduced blood pressure equivalently in young and mature SHR. Aortic medial cell number per unit length was significantly reduced in young, but not mature rats. Expression of pro‐apoptotic protein Bax was significantly reduced in aorta of mature rats. The present study demonstrates that vascular susceptibility to ACEI induced structural remodeling is altered significantly over a relatively short period (15wks) early in adulthood. This may provide a rationale for treating hypertension at an early stage; inasmuch that enhanced vascular remodeling may reduce arterial hypertrophy and stiffening, ultimately leading to improved long‐term protection against hypertensive target organ damage.