Aged SHR Research Articles

Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, β-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

Upregulation of Transient Receptor Potential Canonical Type 3 Channel via AT1R/TGF-β1/Smad2/3 Induces Atrial Fibrosis in Aging and Spontaneously Hypertensive Rats.

Fibroblast proliferation and migration are central in atrial fibrillation (AF) promoting structure remodeling, which is strongly associated with aging and hypertension. Transient receptor potential canonical-3 channel (TRPC3) is a key mediator of cardiac fibrosis and the pathogenesis of AF. Here, we have observed the increased TRPC3 expression that induced atrial fibrosis which possibly is either mediated by the aging process or related to hypertensive progression. In this study, we measured the pathological structure remodeling by H&E staining, Masson staining, and transmission electron microscope (TEM). The protein expression levels of fibrotic biomarkers and TRPC3 were measured by Western blotting with atrial tissues from normotensive Wistar Kyoto rats (WKY 4m-o (4 months old)), old WKY (WKY 24m-o (24 months old)), spontaneously hypertensive rat (SHR 4m-o (4 months old)), and old SHR (SHR 24m-o (24 months old)). To illuminate the molecular mechanism of TRPC3 in atrial fibrosis of aging rats and SHR, we detected the inhibited role of TRPC3 selective blocker ethyl-1-(4-(2,3,3-trichloroacrylamide) phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate,pyrazole-3 (Pyr3) on angiotensin II (Ang II) induced fibrosis in neonatal rat atrial fibroblasts. The pathological examination showed that the extracellular matrix (ECM) and collagen fibrils were markedly increased in atrial tissues from aged and hypertensive rats. The protein expressions of fibrotic biomarkers (collagen I, collagen III, and transforming growth factor-β1 (TGF-β1)) were significantly upregulated in atrial tissues from the WKY 24m-o group, SHR 4m-o group, and SHR 24m-o group compared with the WKY 4m-o group. Meanwhile, the expression level of TRPC3 was significantly upregulated in WKY 24m-o and SHR 4m-o atrial tissues compared to WKY 4m-o rats. In isolated and cultured neonatal rat atrial fibroblasts, Ang II induced the atrial fibroblast migration and proliferation and upregulated the expression levels of TRPC3 and fibrotic biomarkers. TRPC3 selected blocker Pyr3 attenuated the migration and proliferation in neonatal rat atrial fibroblasts. Furthermore, Pyr3 significantly alleviated Ang II-induced upregulation of TRPC3, collagen I, collagen III, and TGF-β1 through the molecular mechanism of the TGF-β/Smad2/3 signaling pathway. Similarly, knocking down TRPC3 using short hairpin RNA (shTRPC3) also attenuated Ang II-induced upregulation of TGF-β1. Pyr3 preconditioning decreased Ang II-induced intracellular Ca2+ transient amplitude elevation. Furthermore, AT1 receptor was involved in Ang II-induced TRPC3 upregulation. Hence, upregulation of TRPC3 in aging and hypertension is involved in an atrial fibrosis process. Inhibition of TRPC3 contributes to reverse Ang II-induced fibrosis. TRPC3 may be a potential therapeutic target for preventing fibrosis in aging and hypertension.

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.

The NO stimulator, Catestatin, improves the Frank–Starling response in normotensive and hypertensive rat hearts

The myocardial response to mechanical stretch (Frank–Starling law) is an important physiological cardiac determinant. Modulated by many endogenous substances, it is impaired in the presence of cardiovascular pathologies and during senescence. Catestatin (CST:hCgA352-372), a 21-amino-acid derivate of Chromogranin A (CgA), displays hypotensive/vasodilatory properties and counteracts excessive systemic and/or intra-cardiac excitatory stimuli (e.g., catecholamines and endothelin-1). CST, produced also by the myocardium, affects the heart by modulating inotropy, lusitropy and the coronary tone through a Nitric Oxide (NO)-dependent mechanism. This study evaluated the putative influence elicited by CST on the Frank–Starling response of normotensive Wistar–Kyoto (WKY) and hypertensive (SHR) hearts by using isolated and Langendorff perfused cardiac preparations. Functional changes were evaluated on aged (18-month-old) WKY rats and SHR which mimic human chronic heart failure (HF). Comparison to WKY rats, SHR showed a reduced Frank–Starling response. In both rat strains, CST administration improved myocardial mechanical response to increased end-diastolic pressures. This effect was mediated by EE/IP3K/NOS/NO/cGMP/PKG, as revealed by specific inhibitors. CST-dependent positive Frank–Starling response is paralleled by an increment in protein S-Nitrosylation. Our data suggested CST as a NO-dependent physiological modulator of the stretch-induced intrinsic regulation of the heart. This may be of particular importance in the aged hypertrophic heart, whose function is impaired because of a reduced systolic performance accompanied by delayed relaxation and increased diastolic stiffness.

Abstract 204: Sodium Sensing Network in Hypertension-Induced Cardiac Hypertrophy

Hypertension is related to alterations of sodium homeostasis that promote abnormal accumulation of water in the intravascular compartment leading to blood pressure elevation. This process can been associated with increased heart mass resulting in cardiac hypertrophy. At cellular level, the concentration and active transport of Na+ is regulated by Na+,K+-ATPase enzyme (NK). In vitro studies have shown a correlation between high blood pressure, NK and SIK activities. Increases in intracellular Na+ are paralleled by elevations in intracellular Ca2+ via NCX1, leading to the activation of SIK1 by a Ca2+/calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK α-subunit and an increase in its catalytic activity. Accordingly, the aim of the present study was to evaluate the cardiac Na+ sensing network changes associated with hypertension in aging hypertensive rats. Systolic and diastolic blood pressures, determined by the tail-cuff method, were significantly higher in 3, 13 and 21-month old SHR than in age-matched WKY. In the SHR, ageing was associated with increases in cardiac mass, gene expression of hypertrophic (α-skeletal muscle actin and β-myosin heavy chain) markers as well as inflammatory cytokine (IL-6). The decreased expression of heart SIK isoforms, SIK1 and SIK3, was associated with downregulation of transcription factors Snail2, Zeb1, MFAT5c and KLF4, in 13 and 21-month old SHR. Cardiac α2-isoform of NK was found to be decreased in 21 month old SHR. Whereas, NKα1, NHE3 and NCX1 showed no differences between age-matched SHR and WKY. Na+-dependent ASCT2 and Na+-independent LAT1 amino acid transporter transcript levels of were significantly higher in 21 month old SHR than in WKY. In the aged SHR, cardiac hypertrophy was associated with dysregulation of several elements of the intracellular Na+ sensing network. Downregulation of SIK1 and transcription factors is correlated with expression of NKα2, suggesting impaired sodium handling. It is proposed that, cardiac hypertrophy is not exclusively the consequence of mechanical stress but also of other factors associated with elevated blood pressure such as abnormal cell sodium homeostasis.Supported by grant PIC/IC/83204/2007.

High salt intake does not produce additional impairment in the coronary artery relaxation of spontaneously hypertensive aged rats

The effect of a salt-based diet on the coronary responsiveness in aged hypertensive rats (SHR) still is unclear. We investigated the effects of high salt intake on the relaxation properties of coronary arteries of aged SHRs. Male SHR (32 week-old) received drinking water (SHR) or 1% NaCl solution (SHR-Salt) for 8 weeks. Isolated coronary segments were subjected to concentration-response curves to acetylcholine (ACh) in the presence or absence of L-NAME (100 μM), enalaprilate (10 μM), losartan (10 μM), and spironolactone (100 μM). Salt intake did not increase blood pressure in old SHRs, but caused ventricular hypertrophy. The endothelium-dependent relaxation in SHRs was lower than in Wistar rats. However, salt intake did not add further impairment. Both enalaprilate and losartan reduced the vasodilator response in coronary arteries from Wistar, but did not affect SHR-salt rats. Conversely, losartan attenuated the impaired ACh relaxation observed in SHR. Spironolactone reduced the relaxation induced by ACh in coronary arteries from Wistar rats but not in SHR. The renin-angiotensin-aldosterone system participates in the impaired coronary relaxation in aged SHR, but does not partake in this deleterious effect under increased salt intake, indicating that age could differentiate the effects of high sodium intake in coronary arteries of SHR.

SEX DIFFERENCE IN BLOOD PRESSURE REGULATION IN AGING SPONTANEOUSLY HYPERTENSIVE RATS

Leptin contributes to sympathetic activation via pro‐opiomelanocortin (POMC) neurons and thereby to increased blood pressure (BP). Whether melanacortin (MC3/4R) receptor contributes to hypertension (HT) in aging SHR is not clear. Our hypothesis was that MC3/4R activation contributes to HT in old SHR. Females and males (16–18 mos, n=6/grp) with MC3/4R antagonist SHU‐9119 (1 nmol/h icv for 7 days). MC3/4R antagonism increased the food intake by 56% in females and 71% in males (females: baseline: 17.1±2 g vs treatment: 26.6±1 g, p&lt;0.001; males: baseline: 25±1.7 g, vs treatment: 43±2 g, p&lt;0.001) and body weight by 18% in females and 23% in males (females: baseline: 217±11 g, vs treatment: 256±6 g, p&lt;0.001; males: baseline: 356±27 g, vs treatment: 438±9 g, p&lt;0.05). MC3/4R antagonism decreased BP (telemetry) and heart rate (HR) in males (BP: baseline: 172±5 vs treatment: 152±4 mmHg, p&lt;0.05; HR: baseline: 321±4 vs treatment: 300±7 bpm, p&lt;0.05), but had no effect in old females (MAP, baseline: 175±13 vs treatment: 172±10 mmHg, p=NS; HR: baseline: 345±9 vs Treatment: 332±9 bpm, p=NS). These results show that while MC3/4R activation contributes to the HT in old male SHR, sympathetic activation in old females is independent of the MC3/4R and suggests that sympathetic activation in aging women may have differential causes than in aging men. Supported by NIH PO1 HL51971, RO1 66072.

Diet Induced Obesity Exacerbates Cardiovascular Dysfunction in the Middle Aged SHR – An Animal Model of Metabolic Syndrome

Background: Obesity and hypertension are two major components of metabolic syndrome that contribute to increased risk of cardiovascular disease. This study aimed to incorporate both hypertension and diet induced obesity in the development of a middle aged animal model of metabolic syndrome. Methods: Male SHR and WKY rats aged 16 weeks were fed either standard rat chow (SC) or given a high-fat-high-carbohydrate (HFHC) diet (23.9% fat, 51.5% carb) for 20 weeks. Fructose (25%) was also administered in the drinking water of HFHC fed rats. Blood pressure, heart rate and oral glucose tolerance was analysed at weeks 0, 4, 8, 12, 16 and 20. Cardiac electrical function was also evaluated using single cell micro electrode physiology. Results: HFHC fed rats gained more body weight and had increased abdominal, retroperitoneal and epididymal fat mass than SC fed rats. Blood pressure was elevated in WKY-HFHC rats (158 ± 6.0 mmHg) but remained stable in SHR-HFHC rats (220.50 ± 8.50 mmHg). There was little change in oral glucose tolerance over the 20 week treatment period. The action potential duration at 90% of repolarisation was prolonged in HFHC-SHR 109.32 ± 8.99 ms; SC-SHR 96.97 ± 15.35 ms; and HFHC-WKY 85.67 ± 6.62 ms compared to SC-WKY 63.83 ± 8.49 ms animals. Conclusion: In addition to obesity, HFHC feeding increases blood pressure in WKY rats and induces alterations in cardiac electrical function in both SHR and WKY middle aged rats. These results suggest that the HFHC middle aged SHR may be a useful animal model for assessing cardiovascular dysfunction in metabolic syndrome and hypertension.

Cardiac benefits of exercise training in aging spontaneously hypertensive rats

To evaluate the effect of low-intensity chronic exercise training (ExT) on blood pressure (BP), as well as the cardiac alterations associated with hypertension in aging hypertensive rats. Male spontaneously hypertensive rats (SHR; 21 months old) and their normotensive control Wistar-Kyoto (WKY) rats were submitted to low-intensity training protocol for 13 weeks. BP, cardiac morphological and morphometric analysis, as well as gene expression of fibrotic and inflammatory factors were analyzed at the end of the training period. ExT reduced BP and heart rate in aged SHR. Left ventricle hypertrophy, collagen volume fraction and wall-to-lumen ratio of myocardium arterioles were also decreased in trained SHR. However, ExT was unable to reverse the either reduced capillary density or the cardiac myocyte hypertrophy observed in SHR as compared with WKY rats. Trained SHR showed higher metalloproteinase-2/tissue inhibitor metalloproteinase-2 (MMP-2/TIMP-2) ratio and lower levels of α-smooth muscle actin, but similar levels of connective tissue growth factor, transforming growth factor beta or IL-1 beta to that of nontrained SHR. Low to moderate-intensity chronic ExT reverses the cardiac alterations associated with hypertension: myocardial arteriole, left ventricle hypertrophy, collagen content and tachycardia. These changes could be consequence or cause of the reduction in BP observed in trained SHR. In addition, ExT does not worsen the underlying inflammatory burden associated with hypertension. Therefore, the data support a beneficial effect of ExT in aging SHR similar to that reported in young or middle-aged individuals, confirming that exercise is a healthy habit that induces cardiac improvements independently of age.

Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/ l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

Down-regulation of fxr nuclear receptor in left ventricle of aging shr: a possible end-stage heart failure bioenergetics adjustment;

Down-regulation of fxr nuclear receptor in left ventricle of aging shr: a possible end-stage heart failure bioenergetics adjustment;

The aging SHR as a model of human heart failure

The aging SHR as a model of human heart failure

Altered responsiveness to angiotensin II in aging SHR causes glomerular hypertension

Altered responsiveness to angiotensin II in aging SHR causes glomerular hypertension

Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats.

In order to investigate SHR hypertrophied heart myocardial vulnerability, superoxide dismutase, a potent scavenger of superoxide anion, was examined in myocardium of SHR and WKY in relation to aging, as well as under doxorubicin (DOX)-induced cardiomyopathy. Superoxide anion production due to doxorubicin administration was also examined histochemically. Superoxide dismutase (SOD), either as Mn-SOD in mitochondria or as CuZn-SOD in cytoplasm, was found to be lower in aged SHR hypertrophied heart than in age-matched WKY heart. Under DOX-induced cardiomyopathy, SOD activity was significantly lower in SHR hypertrophied heart than either control SHR or treated WKY myocardium. Superoxide anion generation, examined morphologically as formazan deposits, was much more intense in SHR myocardium and some degenerative changes were found such formazan-containing cells. These results clearly indicate that the myocardial vulnerability of SHR hypertrophied heart might be a result of a lowered free radical scavenge ability.

In Vivo Evaluation of Left Ventricular Function in Aged SHR by Volume Loading and the Positive Inotropic Effect of Denopamine

The positive inotropic effect of denopamine on the hypertrophied heart was studied in 12-months spontaneously hypertensive rats (12-month-SHR) with and without volume loading. Firstly, the influence of volume loading an cardiac function was studied in 12-month-SHR compared with the age-matched normo-tensive Wistar-Kyoto rats (WKY) and younger WKY (4-month-WKY) as well. Left ventricular end-diastolic pressure (LVEDP) and maximum rate of rise of left ventricular pressure (LV dp/dtmax) were significantly higher in SHR than in WKY and 4-month-WKY. Volume loading (saline, 2 ml/kg/min, i.v., for 10 min) caused a marked increase in LVEDP both in SHR and WKY, but a higher elevation of LVEDP was observed in SHR. LV dp/dtmax, however, was increased in WKY and more markedly in 4-month-WKY, while it was not increased in SHR. Administration of denopamine to SHR at a rate of 1 μg/kg/min, i.v., for 10 min before and during 10 min of volume loading produced a marked increase in LV dp/dtmax and a significant decrease in LVEDP. Under denopamine infusion, volume loading caused an increase in LV dp/dtmax and produced a LVEDP elevation to a similar level to that of WKY. Denopamine at this dose caused no significant effects on heart rate and mean arterial blood pressure, indicating a selective inotropic action. The present study suggests that functional cardiac reserve is reduced in the aged SHR and that denopamine increases the functional reserve of the hypertrophied heart.

Functional and metabolic properties in hearts from aged spontaneously hypertensive rats.

The effect of long-term pressure overload on myocardial functional and metabolic alterations was investigated in hearts from spontaneously hypertensive rats of 16 weeks (young SHR) and 44 weeks (aged SHR) and age matched normotensive Wistar Kyoto strain rats (young WKY, aged WKY). The hearts were perfused by working heart mode and whole heart ischemia was induced by one-way valve. Following 20 min of ischemia, the hearts were reperfused for 30 min. The heart-body weight ratio in both SHR groups was significantly higher than in the respective age-matched WKY groups. Coronary flow relative to heart weight in both SHR groups was significantly lower than that of the respective age-matched WKY during both preischemic and reperfused periods. There was no significant difference in the recovery rate of cardiac output between young and aged WKY, whereas the young and aged SHR revealed significantly less recovery than their respective age-matched WKY. Tissue creatine phosphate and energy charge in both aged groups were significantly lower than in the young groups. These results indicate that long-term pressure overload increases susceptibility to ischemia and decreases the myocardial reserve presumably resulting from relative ischemia, whereas deterioration was minimal in the normotensive aged rat heart.

Effects of α-human atrial natriuretic polypeptide on vascular strips and perfused mesenteric vessels of SHR and WKY

These results show that α-hANP relaxed arterial strips but had no effect on pressor responses obtained in perfused mesenteric vessels. These results are similar to those of atripeptin II, a c-terminal deleted form of α-rANP (Kangawa et al.: BIOCH BIOPHYS RES COMN 121: 585-591, 1984) which relaxes rabbit aortic strips but has no effect on perfused rat hind limb vessels (Oshima et al.: CIRC RES 54: 612-616, 1984), suggesting atrial peptides may have little effects on resistance vessels. The difference observed in aged SHR cannot be explained purely on aging because no change was observed in aged WKY. It might be related to changes induced by the hypertensive state of the SHR.

Inhibition of aging changes by lyophilized Streptococcus faecalis in diet

Effects of dietary Streptococcus faecalis (SF) on aging were evaluated using 1-week, 6-month, and 27-month-old Wistar and SHR rats. SF was lyophilized and added to commercial pellets at 1%. SF inhibited hypertension in aging SHR. SF inhibited aging-related changes in a wide variety of tissues. These changes were a disarray of hepatic cords, portal fibrosis; calcification of cartilage matrix, dominance of chondroitin sulfate B, and keratosulfate over chondroitin sulfates A and C; aortic endothelial damage, segmental thickening, calcification, and chondroid cells in the intima; atrophy of epidermis and appendages, and increase in amorphous dermal matrix; waxy degeneration and atrophy of psoas muscles; increase in lipoperoxides in the serum, liver, and brain; and reduction of cyclic AMP and GMP in the serum. Histological changes of the xyphoid cartilage, aorta, mesenteric artery, liver, dorsal skin, and psoas muscles in 27-month-old SF rats were much less severe than those in controls.

Vascular smooth muscle reactivity to rabbit aorta contracting substance (RCS) and production of prostacyclin-like substance in normotensive and hypertensive rats.

Male SHR of young prehypertensive (5-6 weeks) and mature hypertensive (24-28 weeks) ages and age and sex matched control normotensive Wistar rats (NWR) were studied for aortic smooth muscle contractility in response to rabbit aorta contracting substance (RCS), serotonin, norepinephrine and potassium. Production of prostacyclin (PGI2) in SHR and NWR aortae of these two age groups was also compared. The contractility of young SHR in response to the agonists was all depressed as compared with the matched NWR. With age advances to 24-28 weeks, aortic responsiveness of NWR to all agonists were reduced and the contractility became the same for SHR and NWR except for the response to RCS which was now greater in SHR than in NWR. The PGI2-like substance released from young SHR aortae was the same as that from the matched NWR. The PGI2-like substance in mature aged rat aortae was much higher than that in young rat aortae and the activity in mature SHR was 1.8 times higher than that of the age matched NWR. The increased production of PGI2-like substance in aorta walls of the mature aged SHR may be a compensatory mechanism to reduce the elevated blood pressure.