Dahl Salt-sensitive Hypertensive Rats Research Articles

Central but not peripheral G‐alpha i/o protein inhibition potentiates hypertension in hypertensive Dahl salt‐sensitive rats: a protective role for central G‐alpha i/o proteins

We have shown that intracerebroventricular (i.c.v.) pre‐treatment of rats with pertussis toxin (PTX) prevents the hypotensive and bradycardic responses to the Gαi/o‐protein coupled receptor ligands, nociceptin/orphanin FQ and clonidine. These studies examined whether central Gαi/o protein inhibition alters blood pressure in hypertensive Dahl salt sensitive (DSS) rats.Methods DSS rats were implanted with radio‐telemetry probes and MAP and HR were continuously monitored. After 1‐week, rats were maintained on 8% NaCl chow for 3 weeks then administered either i.c.v. iso‐saline vehicle (N=6), i.c.v. PTX (1 μg) (N=6), or intraperitoneal (i.p.) PTX (15 μg/kg) (N=6).Results DSS rats on 8% NaCl chow showed a significant increase in MAP (normal chow MAP, 130 ± 2 mmHg; 8% NaCl chow, 164 ± 8 mmHg, P<0.05). In hypertensive DSS rats, central PTX significantly increased MAP (day 4 post i.c.v. PTX; MAP Δ +25 ± 2 mmHg, P<0.05). In contrast, peripheral PTX administration produced a significant hypotension (day 4 post i.p. PTX; MAP Δ −58 ± 9 mmHg, P<0.05).Conclusion In hypertensive DSS rats, endogenous central Gαi/o protein pathways participate in reducing the severity of high NaCl‐induced hypertension. This is in contrast to the role of peripheral Gαi/o proteins, which in these (DSS) and previous studies (spontaneously hypertensive rats) play a pathological role in the maintenance of hypertension. Thus, endogenous central vs. peripheral Gαi/o proteins have differential affects on blood pressure control and potentially in the etiology of hypertension. DK43337, HL 071212, P20 RR018766

Estrogen effects on MMP-13 and MMP-14 regulation of left ventricular mass in Dahl salt-induced hypertension

Female Dahl salt-sensitive (DS) rats fed a low-salt diet develop hypertension at 6 months of age. Ovariectomy at 2 months of age accelerates the development of hypertension, and estrogen replacement delays it. Although acute pressure overload induces structural changes in the left ventricle (LV) further effects of gradual hypertension on LV remodeling have not been examined in the DS rat model. The purpose of this study was to test the hypothesis that aging and estrogen loss in hypertensive DS rats are accompanied by changes in LV remodeling. Four groups of DS rats were examined: young intact, middle-aged (MA) intact, MA ovariectomized (MA-OVX), and MA-OVX with 17beta-eestradiol (E(2)) supplementation (MA-OVX+E(2)). Myocardial matrix metalloproteinases (MMPs),tissue inhibitors of metalloproteinases (TIMPs),and extracellular matrix (ECM) proteins were assessed by immunoblotting. Each of the 4 groups comprised 6 animals. Mean (SEM) LV mass was significantly greater in the MA-intact and the MA-OVX groups (1257 [31] mg and 1199 [25] mg, respectively; both, P < 0.05) compared with the young-intact group (697 [6] mg). LV mass in the MA-OVX+E(2) group was significantly lower compared with the MA-intact and MA-OVX groups (both, P < 0.05), suggesting that estrogen may attenuate LV remodeling. Fibronectin and collagen III and IV concentrations increased significantly in the MA-intact and MA-OOVX groups (all, P < 0.05),indicating increased fibrosis. Multiple MMPs also increased in the MA-intact an nd MA-OVX rats, including MMP-3, -7, -99, -113, and -114, and all TIMPs. In contrast, estrogen attenuated fibrosis by increasing MMP-8 concentrations and increasing collagen III fragments. From good-fit regression modeling, MMP-13 and MMP-14 concentrations correlated positively with LV mass for the MA-intact and MA-OVX groups, respectively. Gradual hypertension stimulated ECM turnover by increasing both MMP/TIMP production and ECM degradation. Estrogen loss or gain resulted in a shift in MMP profiles, suggesting that MMP-13 and MMP-14 may be differentially regulated in postmenopausal hypertension.

Closely linked non-additive blood pressure quantitative trait loci

There is enough evidence through linkage and substitution mapping to indicate that rat chromosome 1 harbors multiple blood pressure (BP) quantitative trait loci (QTLs). Of these, BP QTL1b was previously reported from our laboratory using congenic strains derived by introgressing normotensive alleles from the LEW rat onto the genetic background of the hypertensive Dahl salt-sensitive (S) rat. The region spanned by QTL1b is quite large (20.92 Mb), thus requiring further mapping with improved resolution so as to facilitate systematic identification of the underlying genetic determinant(s). Using congenic strains containing the LEW rat chromosomal segments on the Dahl salt-sensitive (S) rat background, further iterations of congenic substrains were constructed and characterized. Collective data obtained from this new iteration of congenic substrains provided evidence for further fragmentation of QTL1b with improved resolution. At least two separate genetic determinants of blood pressure underlie QTL1b. These are within 7.40 Mb and 7.31 Mb and are known as the QTL1b1 region and the QTL1b2 region, respectively. A genetic interaction was detected between the two BP QTLs. Interestingly, five of the previously reported differentially expressed genes located within the newly mapped QTL1b1 region remained differentially expressed. The congenic strain S.LEW(D1Mco36-D1Mco101), which harbors the QTL1b1 region alone but not the QTL1b2 region, serves as a genetic tool for further dissection of the QTL1b1 region and validation of Nr2f2 as a positional candidate gene. Overall, this study represents an intermediary yet obligatory progression towards the identification of genetic elements controlling BP.

Renal Phosphodiesterase 4B Is Activated in the Dahl Salt-Sensitive Rat

Reduced beta-adrenoreceptor signaling is associated with increased sympathoadrenal activity in hypertensive patients and animal models of hypertension. However, the mechanism that accounts for this characteristic decline in beta-adrenergic signaling is unclear. In the present study, we investigated renal phosphodiesterase 4B, which metabolizes cAMP. Immunoblot analysis detected only the phosphodiesterase 4B4 isoform present in kidney tissue from spontaneously hypertensive rats, hypertensive Dahl salt-sensitive (SS) rats, and Dahl salt-resistant rats. The phosphorylated (activated) form of the protein was present at 2-fold greater levels in Dahl SS rats than in spontaneously hypertensive rats and Dahl salt-resistant rats, whereas the unphosphorylated form of the protein was reduced by approximately one half in SS animals. In accord with immunoblot data, rolipram-inhibitable cAMP hydrolyzing activity, a measure of PDE4 activity, was approximately 3-fold greater in kidney cytosolic extracts from SS rats than in extracts from spontaneously hypertensive rats and salt-resistant rats. Phosphodiesterase 4B expression was detected by immunohistochemistry in the renal vasculature, proximal tubules, and distal tubules. These results raise the possibility that increased PDE4 activity, specifically phosphodiesterase 4B4 activity, reduces beta-adrenergic signaling in the kidney and contributes to salt-sensitive hypertension in the Dahl SS rat.

Congenic mapping of a blood pressure QTL region on rat chromosome 10 using the Dahl salt-sensitive rat with introgressed alleles from the Milan normotensive strain

Multiple blood pressure (BP) quantitative trait loci (QTLs) are reported on rat chromosome 10 (RNO10). Of these, QTLs detected by contrasting the genome of the hypertensive Dahl salt-sensitive (S) rat with two different relatively normotensive strains, Lewis (LEW) and the Milan normotensive strain (MNS), are reported. Because the deduced QTL regions of both S vs. LEW and S vs. MNS comparisons are within large genomic segments encompassing more than 2 cM, there was a need to further localize these QTLs and determine whether the QTLs are unique to specific strain comparisons. Previously, the S.MNS QTL1 was mapped to less than 2.6 cM as a differential segment between two congenic strains. In this study, multiple congenic strains spanning the projected interval were studied. The BP effect of each strain was interpreted as the net effect of alleles introgressed within that congenic strain. The results suggest that the MNS alleles within the previously proposed differential segment (D10Rat27-D10Rat24) do not independently lower BP of the S rat. However, another congenic strain, S.MNS(10) x 9, containing introgressed MNS alleles that are outside of the previously proposed differential segment is of interest because (1) it demonstrated a BP-lowering effect, (2) it is contained within a single congenic strain and is not based on the observed effect of a differential segment, and, more importantly, (3) it overlaps with the previously identified S.LEW BP QTL region. Identification of the same QTL affecting BP in multiple rat strains will provide further support for the QTL's involvement and importance in human essential hypertension.

Assessment of Mitochondrial DNA Polymorphisms in Salt-Sensitive Hypertension in Dahl Salt-Sensitive Rats

The Dahl salt-sensitive (DS) rat is the most prevalently used animal model of salt-sensitive hypertension. The purpose of the present study was to test the hypothesis that mitochondrial DNA (mtDNA) polymorphisms influence blood pressure in DS rats. We produced two strains of female F1 rats, one from female DS and male Lewis rats (DL) and the other from Lewis female and DS male rats (LD). These two strains had the same autosomal genetic background, but their mitochondria had different origins. The DL and LD rats had DS and Lewis mitochondria, respectively. A high-salt diet was started at 4 weeks of age. Radiotelemetry devices were implanted into the lower abdominal aorta of these F1 rats at 9 weeks of age. Blood pressure was monitored for 24 h at 11, 12, 13, 14, and 19 weeks of age. No significant differences were observed in blood pressure levels between the strains. Although more than 100 polymorphisms were detected between DS and Lewis rats, it is unlikely that polymorphisms in mtDNA contribute to hypertension in DS rats. Moreover, we found no difference between DS and Lewis rats in the mtDNA copy number in the kidneys, the liver, and the ventricles of the heart before and after salt loading. Thus, it is unlikely that mitochondrial dysfunction due to high blood pressure exacerbated target organ damage. Intriguingly, the time course of body weight gain differed significantly between DL and LD F1 rats, suggesting the influence of mitochondrial polymorphisms on body composition.

Congenic expression of tissue inhibitor of metalloproteinase in Dahl-salt sensitive hypertensive rats is associated with reduced LV hypertrophy

Although congenic translocation of a segment from chromosome 10 from Lewis rat, containing an extracellular proteinase inhibitor gene, decreased blood pressure in Dahl-salt sensitive (DSS) rats, the relationship between the levels of matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and cardiac function was unclear. In this study we investigated the cardiac effects of congenic translocation of a segment from chromosome 10 from Lewis rat, containing an extracellular proteinase inhibitor gene, in Dahl-salt sensitive rats. To test the hypothesis that left ventricular (LV) hypertrophy in DSS rats was due to high MMP and low TIMP levels and the decrease in blood pressure in congenic rats was associated with increase in proteinase inhibitor expression, cardiac function and levels of MMP and TIMP were determined in 16 weeks male DSS (D), Lewis (L) and congenic (CL-10) rats. Cardiac function was assessed by electrocardiography, echocardiography and a Millar catheter in LV cavity. LV MMP and TIMP levels were measured by Q-RT-PCR and Western blot analyses. In L, D and CL-10 rats, heart weight/body weight (g/g) were 3.73 ± 0.06, 4.45 ± 0.04 and 3.35 ± 0.05 × 10−3, respectively, suggesting significant (p < 0.05) LV hypertrophy (LVH) in D group. The ST duration was longer in D group compared with L group, suggesting coronary vasospasm, but normalized in CL-10 rats. The fractional shortening and ejection fraction were decreased in D group as compared with L group, but normalized in CL-10 groups. LV diameter was increased in D group as compared to L group, but normalized in CL-10 groups. The levels of MMP-9 were higher and TIMP were lower in D as compared to L groups, but normalized in CL-10 rats. Compared with control non-congenic Dahl rats, congenic rats exhibited lower blood pressure, amelioration of LV remodelling and dysfunction, as well as coronary abnormalities. In addition, congenic animals exhibited reduced myocardial expression of MMP-9, but increased expression of MMP-2 and TIMP-4 compared to non congenic animals. We concluded that the congenic transfer of TIMP ameliorated LV hypertrophy and cardiac dysfunction.

Suppression of Cardiomyocyte Hypertrophy by Inhibition of the Ubiquitin-Proteasome System

Inhibitors of the proteasome interfere with transcriptional regulation of growth signaling pathways and block cell cycle progression of mitotic cells. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of postmitotic cardiomyocytes. To test this hypothesis, we analyzed the effects of proteasome inhibition on hypertrophic growth of neonatal rat cardiomyocytes. Partial inhibition of the proteasome effectively suppressed cardiomyocyte hypertrophy as determined by reduced cell size, inhibition of hypertrophy-mediated induction of RNA and protein synthesis, reduced expression of several hypertrophic marker genes, and diminished transcriptional activation of the BNP promotor. Importantly, suppression of hypertrophic growth was independent of the hypertrophic agonist used. Expressional profiling and subsequent Western blot and kinase assays revealed that proteasome inhibition induced a cellular stress response with reduced expression of conserved growth signaling mediators and impaired G1/S phase transition of cardiomyocytes. In hypertensive Dahl-salt sensitive rats, inhibition of the proteasome with low doses of the FDA approved proteasome inhibitor Velcade significantly reduced hypertrophic heart growth. Our data provide important insight into the suppressive effects of proteasome inhibitors on hypertrophic growth of cardiomyocytes and establish low-dose proteasome inhibition as a new and broad-spectrum approach to interfere with cardiac hypertrophy.

Abstract 670: Immediate And Sustained Blood Pressure Lowering by CRF-receptor Stimulation: A Novel Approach To Antihypertensive Therapy?

Background: Recently, novel corticotropin-releasing factor (CRF)-related peptides, named urocortin I (UcnI), UcnII, and UcnIII were described. Available data suggest that the Ucns are part of a peripheral CRF system modulating cardiovascular function and mediating cardiovascular responses to stress. Blood pressure (BP) lowering effects have been described after administration of UcnI. However, no data are available on effects of UcnII on BP in an animal model of systemic arterial hypertension. Methods: Experiments were performed in Dahl salt-sensitive (DSS) and salt-resistant rats (DSR, control). Animals were fed a diet containing 4% NaCl (high salt) to induce arterial hypertension in DSS rats. At the end of week 2 of high salt diet, both DSS and DSR rats were randomly assigned to i.p. injections of either UcnII (2.5 μg/kg body weight) or vehicle b.i.d. for five weeks. Animals underwent repetitive tail cuff BP measurements at baseline (prior to first injection), at 5 and 15 minutes after the first injection and at week 1, 2, and 5 of b.i.d. treatment. At week 5 animals were sacrificed to determine heart weight /body weight ratio. Results: Systolic BP (SBP, mmHg) and heart rate (HR, min −1 ) are given in the following table as mean ± SD (n=10 per group). Conclusions: In hypertensive DSS rats, acute CRF-receptor stimulation by UcnII immediately lowered BP to the range observed in DSR rats. Compared to vehicle-treated DSS rats, sustained BP reduction was observed with further chronic administration of UcnII. No severe reflex tachycardia was observed after administration of UcnII. Thus, CRF-receptor stimulation might represent a novel approach to the treatment of arterial hypertension.

Abstract 2523: Azelnidipine Enhances Beneficial Effects of Olmesartan on Left Ventricular Remodeling During the Development of Hypertension-induced Heart Failure

Objective: This work was undertaken to investigate the comparative effect of angiotensin II type 1 receptor blocker (ARB) and a combination of ARB and calcium channel blocker (CCB) on left ventricular (LV) remodeling during the development of hypertensive heart failure (H-HF). Methods and Results: We treated 8% salt-loaded Dahl salt-sensitive hypertensive rats (n = 10 for each group) with vehicle, hydralazine (5 mg/kg/d), olmesartan (OLM, 5 mg/kg/d), or combined OLM and azelnidipine (AZE, 2mg/kg/d) for 8 weeks. The rats fed 0.3% salt served as age-matched controls. The abundance of Cat mRNAs and proteins were localized in cardiac myocytes (CMCs), and Cat-dependent activities were increased by 4.1-fold in the LV of H-HF rats (n = 8, P&lt; 0.001) and were reduced by OLM treatment. OLM suppressed the elastic lamina degradation concomitant with decreased local Cat S expression in intracoronary smooth muscle cells (SMCs) and restored the balance of elastin to collagen in the LV tissue of H-HF rats (H-HF 4.6 ± 0.9% vs. OLM 15.5 ± 2.1% elastin content/collagen content (%), n = 6, P&lt; 0.0±1; control 22±2.1%). OLM suppressed not only macrophage infiltration but also levels of NADPH oxidase components (p22 phox , gp91 phox , and p47 phox ) concomitant with decreased NADPH activity and O2- production in LV tissues of H-HF rats. Along with its comparable anti-inflammatory effect, add-on AZE further improved all of these parameter changes by OLM. Furthermore, combination therapy significantly enhanced the improvement of LV fibrosis, hypertrophy, stiffness, and dysfunction by OLM. In vitro, H 2 O 2 stimulated Cat S mRNA and protein expression and activity, and these increases were abolished by pretreatment with the antioxidants such as MnTmPyp (50 μmol/L) and N-acetylcysteine (5 mmol/L) as well as a NADPH oxidase inhibitor apocynin (100 μmol/L) in culture CMCs, SMCs, and macrophages (n = 6, P&lt; 0.01). Conclusions: OLM and a combination of OLM and AZE exerted cardioprotective effects in hypertensive HF, via elastolytic Cat activation inhibition by the reduction of NADPH oxidase-dependent superoxide anion production. AZE enhanced the cardioprotective effects of OLM. Thus, the combination of ARB with CBB is a promising potential therapeutic strategy for H-HF.

Abstract 3294: Superoxide-Dependent Cathepsin Activation System is Associated with Hypertensive Myocardial Remodeling and Represents a Target for Angiotesin II Type 1 Receptor Blocker Therapy

Objective: Cathepsin (Cat) activation contributes to tissue remodeling, and increased Cat activity and superoxide production has been observed in the left ventricle (LV) during heart failure (HF). In the present study we tested the hypothesis that superoxide-dependnet Cat activation inhibition prevents LV remodeling and dysfunction in the development of HF associated with hypertension. Methods and Results: We treated 8% salt-loaded Dahl salt-sensitive hypertensive rats (n=10 for each group) with vehicle hydralazine (5 mg/kg/d) E64d (a synthesis Cat inhibitor, 10 mg/kg/d), or olmesartan (OLM, 5 mg/kg/d) for 8 weeks. The rats fed 0.3% salt served as age-matched controls. The abundance of Cat mRNAs and proteins localized in cardiac myocytes (CMCs) and Cat-dependent activities were increased in the LV of HF rats and/or humans, and were reduced by OLM treatment. OLM suppressed the elastic lamina degradation by 56% (n=6, p&lt;0.01) concomitant with decreased local Cat S expression in intracoronary smooth muscle cells (SMCs) and restored the balance of elastin to collagen in the LV tissue of HF rats (HF 4.6 ± 0.9% vs. OLM 15.5 ± 2.1% elastin content/collagen content (%), n=6, P=0.031; control 22 ± 2.1%). Furthermore, OLM suppressed not only angiotensin converting enzyme and angiotensin II type 1 receptor (AT 1 R) and macrophage infiltration but also levels of NADPH oxidase components (p22 phox , gp91 phox , and p47 phox) concomitant with decreased NADPH activity and O 2 − production in LV tissues of HF rats and humans. These changes were accompanied by improved cardiac fibrosis, stiffness, and dysfunction. Interestingly, LV remodeling and dysfunction were partially improved by treatment with E64d. In vitro, H 2 O 2 stimulated Cat S mRNA and protein expression and activity, and these increases were abolished by pretreatment with MnTmPyp (50 μ mol/L) and N-acetylcysteine (5 mmol/L) as well as apocynin (100 μ mol/L) in culture CMCs (n=6, p&lt;0.01). Conclusions: This is the first report showing that Cats are likely to trigger and promote LV remodeling, and that AT 1 R inhibition exerts inhibitory effect on Cat activation system by the inhibition of NADPH oxidase-dependent superoxide anion production, leading to the prevention of cardiac remodeling and dysfunction.

Novel Mechanism and Role of Angiotensin II–Induced Vascular Endothelial Injury in Hypertensive Diastolic Heart Failure

The mechanism and role of angiotensin II-induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure. We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1(-/-) mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1(-/-) mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure. AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.

Effects of Aldosterone and Angiotensin II Receptor Blockade on Cardiac Angiotensinogen and Angiotensin-Converting Enzyme 2 Expression in Dahl Salt-Sensitive Hypertensive Rats

We previously reported that a high-sodium diet activates the local renin-angiotensin-aldosterone system (RAAS) in cardiovascular tissues of Dahl salt-sensitive hypertensive (DS) rats. Angiotensin-converting enzyme 2 (ACE2) is a novel regulator of blood pressure (BP) and cardiac function. The effect of blockade of aldosterone or angiotensin II (Ang II) on cardiac angiotensinogen and ACE2 in DS rats is unknown. The BP, plasma renin activity (PRA), plasma aldosterone concentration (PAC), heart weight, endothelium-dependent relaxation (EDR), and messenger RNA (mRNA) levels of collagen III, angiotensinogen, ACE, and ACE2 in the heart were measured in DS rats and in Dahl salt-resistant (DR) rats fed high or low salt diets. The rats were treated orally with or without eplerenone (100 mg/kg/d), candesartan (10 mg/kg/d), or both drugs combined for 8 weeks. A high salt diet increased BP (140%), heart/body weight (132%), and collagen III mRNA levels (146%) and decreased PRA and PAC concomitant with increased expression of cardiac angiotensinogen mRNA and decreased mRNA levels of ACE2 in DS rats. Eplerenone or candesartan significantly decreased the systolic BP from 240 +/- 5 mm Hg to 164 +/- 4 mm Hg or to 172 +/- 10 mm Hg, respectively (P < .05). Eplerenone or candesartan partially improved heart/body weight and cardiac fibrosis, improved EDR and decreased cardiac ACE and angiotensinogen mRNA levels in DS rats. Candesartan increased ACE2 mRNA levels in the heart. Combination therapy normalized BP and further improved cardiac hypertrophy, fibrosis, and EDR. In DS rats, blockade of aldosterone or Ang II protects cardiac hypertrophy and fibrosis by inactivation of the local RAAS in the heart.

Role of Xanthine Oxidoreductase in the Reversal of Diastolic Heart Failure by Candesartan in the Salt-Sensitive Hypertensive Rat

The role of angiotensin II and reactive oxygen species in the exacerbation of diastolic heart failure is unknown. We examined the therapeutic effect of angiotensin blockade on hypertensive diastolic heart failure, focusing on the role of xanthine oxidoreductase and reduced nicotinamide-adenine dinucleotide phosphate oxidase, major enzymes producing reactive oxygen species. Dahl salt-sensitive hypertensive rats (DS rats) with established diastolic heart failure were given vehicle, candesartan (an angiotensin II receptor subtype 1 receptor blocker), oxypurinol (a xanthine oxidoreductase inhibitor), apocynin (a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor), or hydralazine (a vasodilator), and their therapeutic effects on diastolic heart failure were compared. Candesartan treatment of DS rats with established diastolic heart failure reversed cardiac remodeling, improved cardiac relaxation abnormality, and prolonged survival, being accompanied by the attenuation of the increase in cardiac superoxide, reduced nicotinamide-adenine dinucleotide phosphate oxidase, and xanthine oxidoreductase activities. Thus, the beneficial effect of candesartan in DS rats appears to be mediated by the inhibition of cardiac reactive oxygen species. Cardiac xanthine oxidoreductase inhibition with oxypurinol significantly reduced cardiac superoxide, prevented the progression of cardiac remodeling, and delayed the mortality in DS rats. Apocynin, which significantly inhibited cardiac reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, prevented the exacerbation of diastolic heart failure more than hydralazine. However, compared with candesartan or oxypurinol, apocynin did not improve cardiac reactive oxygen species, remodeling, and function in DS rats. In conclusion, candesartan slowed the exacerbation of hypertensive diastolic heart failure in DS rats by causing reverse cardiac remodeling. Cardiac xanthine oxidoreductase contributed to these beneficial effects of candesartan.

Cardioprotective effect of angiotensin II type 1 receptor antagonist associated with bradykinin-endothelial nitric oxide synthase and oxidative stress in Dahl salt-sensitive hypertensive rats

The interactions between eNOS or oxidative stress and bradykinin under long-term treatment of angiotensin II type 1 receptor antagonists (ATRA) remain unknown. To elucidate the molecular mechanisms of the cardioprotective effect of ATRA, we evaluated whether valsartan affects the bradykinin-eNOS and nicotinamide adenine dinucleotide (NAD(P)H) oxidase pathway. After 5 weeks of feeding an 8% NaCl diet to 6-week-old Dahl salt-sensitive hypertensive (DS) rats, a distinct stage of concentric left ventricular hypertrophy (LVH) was noted. Six-week-old DS rats were treated with one of the following drug combinations for 5 weeks until the onset of LVH: vehicle; bradykinin B2 receptor antagonist FR172,357 alone; high-dose hydralazine; low-dose hydralazine; high-dose valsartan; low-dose valsartan; high and low-dose valsartan plus FR172,357. Age-matched Dahl salt-resistant rats fed the same diet served as controls. eNOS expression and activity, which was decreased in hypertrophy, was increased by high or low-dose valsartan, but not by high and low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. The increased expression of NAD(P)H oxidase p22phox, p47phox, p67phox, and gp91phox in DS rats was suppressed by high or low-dose valsartan, but not by high or low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. Valsartan effectively inhibited vascular lesion formation and suppressed the expression of transforming growth factor-beta1, connective tissue growth factor, and type I collagen, but not valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. These findings suggest that valsartan may have cardioprotective effects in this model, partly associated with the bradykinin-eNOS and oxidative stress pathway.

Beneficial Effects of the Combination of Nifedipine and Losartan in Hypertensive Dahl Salt-Sensitive Rats

The antihypertensive and organ-protective effects of the combination of the angiotensin II type 1 receptor blocker losartan and the calcium channel blocker nifedipine were examined in Dahl salt-sensitive rats. The rats fed with a high-salt diet developed hypertension accompanied by aorta and heart hypertrophy, and impaired renal function. The animals were treated with losartan (30 mg/kg/day), nifedipine (7.8 mg/kg/day) or with a combination of both drugs for 8 weeks. At the end of the study systolic blood pressure, kidney function, organ weight, and mRNA expression were investigated. Losartan reduced significantly the systolic blood pressure as well as the aorta and left ventricular hypertrophy. Nifedipine and its combination with losartan had similar effects on the systolic blood pressure, aorta and left ventricular hypertrophy but only the combination treatment reduced the expression of transforming growth factor-beta1 in aorta and brain natriuretic peptide in left ventricle significantly. Nifedipine and the combination therapy reduced proteinuria and improved urine creatinine excretion. The expression of collagen III and IV in the kidney was significantly reduced by the combination therapy. These results indicate that although losartan and nifedipine were effective in lowering blood pressure and showed moderate organ protection, additional benefits can be expected by combination therapy with both compounds.

Albuminuria, Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Monocyte Chemoattractant Protein-1 in the Renal Tubules of Hypertensive Dahl Salt-Sensitive Rats

In chronic renal diseases, experimental and human data suggest that excess albumin filtered through the glomerular capillary barrier is over-reabsorbed by proximal tubular cells, thereby activating these cells and upregulating the expression of chemokines. On the other hand, a high-salt diet has been shown to induce proteinuria in hypertensive Dahl salt-sensitive (DSS) rats, accompanied with the expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney. In the current study, we therefore examined albuminuria and the expressions of NADPH oxidase and monocyte chemoattractant protein-1 (MCP-1) in the renal tubular cells in hypertensive DSS rats, as well as the effects of the antioxidant N-acetylcysteine (NAC) on each of these parameters. DSS rats were fed a normal-salt diet (0.24% NaCl), a high-salt diet (8% NaCl), or a high-salt diet plus NAC supplementation (15 mg/mL drinking water) for 4 weeks. The high-salt diet provoked an increase in glomerular injuries accompanied with albuminuria and in urinary H2O2 and MCP-1 excretion. Immunohistochemical analysis showed the prominent expression of MCP-1 in the dilated tubular cells, where the NADPH oxidase subunit p47phox was also expressed. The current results suggest that albuminuria caused expression of NADPH oxidase and MCP-1 in the dilated renal tubules, resulting in interstitial inflammation and migration of mononuclear cells in DSS rats, because blockade of albuminuria by NAC counteracted the p47phox and MCP-1 expression.

High sucrose diet causes increased left ventricular dysfunction and mortality in hypertensive Dahl salt sensitive rats.

Chronic hypertension (HTN) often leads to left ventricular hypertrophy (LVH), LV dysfunction, and death. The guidelines for carbohydrate (CHO) intake, specifically simple sugars, for hypertensive individuals are unclear. We hypothesized that high simple sugar diets will accelerate LV dysfunction and death compared to high starch and high fat diets fed to hypertensive Dahl salt sensitive rats. Four diets were used: Complex CHO (CC; 70% starch, 10% fat, 20% protein by calories), High Fat (FAT; 20% CHO, 60% fat, 20% protein), High Fructose (FRU; 70% fructose, 10% fat, 20% protein), and High Sucrose (SUC; 70% sucrose, 10% fat, 20% protein). HTN was initiated by adding 6% NaCl (+S) to the chow of half the rats within each diet (n=8–11/group). All rats fed a high salt diet developed similar levels of HTN, and demonstrated increased LVH compared to rats fed low salt diets. Echocardiography demonstrated that rats in the SUC+S group had decreased LV performance as evidenced by decreased ejection fraction compared to all other hypertensive groups. All rats fed a low salt diet survived 8 weeks of treatment, while slight mortality was observed in the CC+S, FAT+S, and FRU+S groups. Conversely, significant mortality was observed in the SUC+S group (44%). These results show that a high sucrose diet consumed during HTN increases LV dysfunction and mortality compared to either a high fat, high starch, or a high fructose diet.

Suppression of Catechol-O-Methyltransferase Activity through Blunting of .ALPHA.2-Adrenoceptor Can Explain Hypertension in Dahl Salt-Sensitive Rats

Catecholamines have been reported to be involved in the development of salt-sensitive hypertension. We investigated the relation between catechol-O-methyltransferase (COMT) and salt-sensitivity. In the first experiment, Dahl salt-sensitive (DS) rats were given a normal-salt (NS), high-salt (HS), or HS+hydralazine (80 mg/l water) diet for 4 or 13 weeks, and Dahl salt-resistant (DR) rats were given a NS or HS diet. COMT activities in both the kidneys and liver and urinary norepinephrine (NE) and dopamine (DA) excretion were measured. In the second experiment, HepG2 cells were used to investigate the role of NE in regulating COMT activity. In the third experiment, we investigated the reactivity of pre- and postsynaptic alpha(2)-adrenoceptor (AR) in DS rats. HS loading significantly suppressed the activities of membrane-bound COMT (MB-COMT) and, consistent with this finding, increased the urinary NE level in DS rats, but not in DR rats. Hydralazine did not restore the MB-COMT activities, which suggested that HS loading rather than elevated blood pressure suppressed the MB-COMT activities. The in vitro experiment using HepG2 cells revealed that NE increased the MB-COMT activity via the alpha(2)-AR. However, both the pre- and postsynaptic alpha(2)-AR reactivity was decreased by HS loading in DS rats. In conclusion, HS intake suppresses the MB-COMT activities in DS rats, presumably by blunting alpha(2)-AR signaling. The suppression of MB-COMT activities, consequent decrease in degradation of NE, and increase in NE release by blunting of alpha(2)-AR function may be involved in the development of salt-sensitive hypertension in DS rats, in whom DA-dependent natriuresis may be suppressed.

The Monocyte Chemotactic Protein-1 Gene May Contribute to Hypertension in Dahl Salt-Sensitive Rats

In a previous study, we performed a genome-wide quantitative trait loci (QTLs) analysis for blood pressure using F2 rats derived from Dahl salt-sensitive (DS) and Lewis (LEW) rats and identified two QTLs that influenced blood pressure levels. Although we determined that one of the causative genes in the chromosome (Ch) 1 region seemed to be Klk1, we did not perform detailed analyses on the Ch10 QTL region. The purpose of the present study was to identify candidate genes that influence blood pressure in the Ch10 QTL region. Using microarray analysis, we compiled a list of the genes that are differentially expressed between the two strains and that were localized to the Ch10 QTL region. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis identified that, while the expression levels of Ccl2 mRNA were not different between the kidneys of DS and LEW rats fed a normal diet, those in DS were 10-fold higher than those in LEW under a high-salt diet. Although the promoter reporter assay failed to identify causative nucleotide changes that led to the differential expression, monocyte chemotactic protein-1 (MCP-1) release from isolated monocytes were significantly higher in DS than in LEW. Intriguingly, this Ch10 QTL for blood pressure was also a possible QTL for urinary albumin excretion. Since Ccl2 is well known to be involved in various types of renal injury, it is likely that a higher expression of Ccl2 might aggravate macrophage infiltration, which in turn could aggravate tubulointerstitial injury, and thereby accelerate salt-sensitive hypertension. Thus, Ccl2 appears to be a interesting candidate gene for salt-sensitive hypertension in DS.