Dahl-S Rats Research Articles

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension

Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

The T8590C Polymorphism of CYP4A11 and 20-Hydroxyeicosatetraenoic Acid in Essential Hypertension

A role for a deficit in transport actions of 20-hydroxyeicosatetraenoic acid (20-HETE) in hypertension is supported by the following: (1) diminished renal 20-HETE in Dahl-S rats; (2) altered salt- and furosemide-induced 20-HETE responses in salt-sensitive hypertensive subjects; and (3) increased population risk for hypertension in C allele carriers of the T8590C polymorphism of CYP4A11, which encodes an enzyme with reduced catalytic activity. We determined T8590C genotypes in 32 hypertensive subjects, 25 of whom were phenotyped for salt sensitivity of blood pressure and insulin sensitivity. Urine 20-HETE was lowest in insulin-resistant, salt-sensitive subjects (F=5.56; P<0.02). Genotypes were 13 TT, 2 CC, and 17 CT. C allele frequency was 32.8% (blacks: 38.9%; whites: 25.0%). C carriers (CC+CT) and TT subjects were similarly distributed among salt- and insulin-sensitivity phenotypes. C carriers had higher diastolic blood pressures and aldosterone:renin and waist:hip ratios but lower furosemide-induced fractional excretions of Na and K than TT. The T8590C genotype did not relate to sodium balance or pressure natriuresis. However, C carriers, compared with TT, had diminished 20-HETE responses to salt loading after adjustment for serum insulin concentration and resetting of the negative relationship between serum insulin and urine 20-HETE to a 1-microg/h lower level of 20-HETE. The effect of C was insulin independent and equipotent to 18 microU/mL of insulin (Delta20-HETE= 2.84-0.054xinsulin-0.98xC; r(2)=0.53; F=11.1; P<0.001). Hence, genetic (T8590C) and environmental (insulin) factors impair 20-HETE responses to salt in human hypertension. We propose that genotype analyses with sufficient homozygous CC will establish definitive relationships among 20-HETE, salt sensitivity of blood pressure, and insulin resistance.

Effects of Mannooligosaccharides from Coffee Mannan on Blood Pressure in Dahl Salt-Sensitive Rats

Dahl salt-sensitive (Dahl-S) rats, serving as a model of hereditary hypertension, were used to examine the effect of mannooligosaccharides (MOS) on blood pressure. Dahl-S rats were induced to develop hypertension by administering them with a 1.25% salt solution ad libitum. In a 10-wk experimental period, the Dahl-S control and MOS groups developed and maintained significantly higher blood pressure than the Dahl salt-resistant normal control group. The MOS group showed a significantly lower blood pressure than the Dahl-S control group after 5-wk of treatment (p<0.05). In addition, the serum aldosterone level of the MOS group significantly decreased (p<0.05). The findings of this study using a model of hypertensive rats suggest that MOS are able to suppress an elevation in blood pressure.

Chimeric DNA–RNA hammerhead ribozyme targeting transforming growth factor-β1 mRNA ameliorates renal injury in hypertensive rats

Transforming growth factor (TGF)-beta is a critical factor in the progression of renal injury, regardless of the primary etiology. Such injury is characterized by glomerular sclerosis and tubulointerstitial fibrosis. To develop a ribozyme-based therapy for progressive renal diseases, we examined the effects of chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta1 mRNA on glomerulosclerosis in salt-loaded, stroke-prone spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl (Dahl-S) rats. The chimeric DNA-RNA ribozyme to TGF-beta1 was delivered by polyethylenimine to cultured mesangial cells from SHR-SP in vitro and to glomeruli in SHR-SP in vivo. The chimeric ribozyme reduced expression of TGF-beta1 mRNA and protein, which was accompanied by inhibition of expression of extracellular matrix molecules such as fibronectin and collagen type I in mesangial cells from SHR-SP in vitro. One intraperitoneal injection of 200 microg of chimeric DNA-RNA ribozyme to TGF-beta1 in vivo markedly ameliorated thickening of capillary artery walls and glomerulosclerosis in salt-loaded SHR-SP and Dahl-S rats without a reduction in blood pressure. The chimeric ribozyme reduced expression of TGF-beta1 and connective tissue growth factor (CTGF) mRNAs in renal cortex in salt-loaded Dahl-S rats. Chimeric ribozyme to TGF-beta1 significantly reduced levels of protein in urine in the Dahl-S rats. These results suggest that chimeric DNA-RNA ribozyme to TGF-beta1 may be useful as a gene therapy for progressive tissue injury in a wide variety of renal diseases, including hypertensive nephrosclerosis.

Distinct rapid and slow phases of salt-induced hypertension in Dahl salt-sensitive rats

To test the hypothesis that Dahl salt-sensitive (Dahl-S) rats exhibit distinct and separable phases of salt sensitivity. Blood pressure (BP) telemetry was used to describe the detailed time course of salt-induced hypertension in Dahl-S rats and in hybrid rats derived from Dahl-S and Dahl salt-resistant strains. Switching to a high salt (4% NaCl) diet led to a biphasic increase in BP. Phase-1 reached a plateau in 4 days whereas phase-2 progressed slowly over the subsequent 5 weeks. In hybrid rats, phase-1 was present in each rat whereas phase-2 was absent in many individuals. A correlation of the amplitude of the first and second phases was of borderline significance in Dahl-S rats (P = 0.053, R2 = 0.44, n = 9) but was clearly significant in hybrid rats (P < 0.0001, R2 = 0.78, n = 22). Increases in BP were reversible following 1 week of high salt but progressively less so after 4 and 7 weeks. Estimation of the chronic pressure-natriuresis relationship suggests that phase-1 is attributable to a reduced slope of this relationship. In contrast, phase-2 corresponds with a further reduction in slope and a progressive and irreversible resetting of the relationship to higher BP levels. Two phases of salt sensitivity coexist and provide distinct contributions to salt-induced hypertension in Dahl-S rats. Our data also suggest that short-term measures of salt-sensitivity may be predictive of the effect of salt on the eventual progression of salt-induced hypertension.

High Salt Intake Decreases EET Excretion in Stroke‐Prone Spontaneously Hypertensive Rats (SHRSP)

Epoxyeicosatrienoic acids (EETs) have renal vasodilatory, natriuretic and diuretic activities. High salt intake stimulates production of EETs and diminished EETs formation may be related to the development of salt-sensitive hypertension in Dahl-S rats. We evaluated changes in EETs production in SHRSP as the development of vascular lesions is highly dependent on salt intake in these animals. Urine was collected from male Wistar-Kyoto rats (WKY) and SHRSP (Charles River, 6 week old) before (7 weeks old), and 10 days after, placement on 1% NaCl (HS) drinking solution. Urinary EETs and dihydroxyeicosatrienioc acids (DHETs), metabolites of EETs were quantitated by GCMS. Urine volume (UV) was lower and excretion of EETs+DHETs was higher in SHRSP than WKY under normal salt conditions (NS, 0.4% NaCl). WKY showed an increase in UV, but no change in EETs or DHETs excretion in response to HS intake. In contrast, excretion of EETs and DHETs was markedly decreased in SHRSP in response to HS intake. These results demonstrate a major alteration in EETs formation in response to HS intake in SHRSP. Whether this defect in EETs formation contributes to the development of pathology in SHRSP remains to be investigated.

Development of Gene Silencing Pyrrole-Imidazole Polyamide Targeting the TGF-β1 Promoter for Treatment of Progressive Renal Diseases

Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to the minor groove of DNA. A Py-Im polyamide that targets rat TGF-beta1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-beta1 gene, recombinant TGF-beta1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-beta1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-beta1 promoter activity and expression of TGF-beta1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-beta1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-beta1 will be a novel gene-silencing agent for the TGF-beta1-associated diseases, including progressive renal diseases.

Commentary: Role of salt intake in the development of high blood pressure

Louis Dahl’s famous graph in 1960 showing a positive linear relationship between prevalence of hypertension and mean salt intake across five population groups caught the imagination of the blood pressure research community, and it remains influential to this day. It led to an intensification of research on the role of salt in hypertension spanning anthropology, epidemiology, animal studies, studies of mechanisms and clinical trials. In his 1960 paper, Dahl encompasses much of the basic thinking underpinning current-day public health efforts to reduce sodium consumption in the population. First, he notes that daily intakes of salt among Americans and other populations were well in excess of physiological need ~1 g salt/day (17 mmol sodium) compared with the 10 g (170 mmol sodium) or more being consumed on average by a white American man, and.26 g (440 mmol sodium) by northern Japanese farmers (rates of hypertension and stroke were exceptionally high in northern Japan at that time). He remarks that an individual’s dietary salt intake is highly variable and difficult to measure (noting that 24 h urinary excretion was the preferred method); also that salt appetite in humans is induced rather than innate, such that people on a low sodium diet (in some cases as low as a few mmol sodium/day) could rapidly adapt, and furthermore suffered no ill effects. In addition, his animal experiments (and those of Meneely et al. 1 ) had shown that hypertension in rats could be induced by sodium ingestion, in a dose-dependent way over a prolonged period. Though not an epidemiologist by training, he understood and expounded the concept that an environmental risk factor (salt intake) could increase the risk of disease (high blood pressure) in a group, while acknowledging that the risk for any given individual also depended on other factors, including genetic susceptibility. Therefore, the experiment of choice was to examine disease occurrence (rates of hypertension) in different populations with wide variations in exposure (salt intake, measured by 24 h urinary sodium excretion), rather than to compare blood pressures and sodium intakes of individuals within a population. Later, through a series of breeding experiments Dahl expanded on the concept of gene–environment interaction with respect to an animal model of salt and hypertension; he in-bred a strain of rats (Dahl-S rats), which were ‘sensitive’ to salt intake—fed a diet high in salt, the rats would go on to develop hypertension and stroke. In contrast Dahl-R rats (‘resistant’) could tolerate high salt intake without developing hypertension. 2 In his 1960 paper, Dahl postulated that humans would show a range of responses to a high salt intake, but that at the group level, hypertension would be uncommon below an intake of 4–5 g salt/day (68–85 mmol sodium/day). Other authors subsequently extended Dahl’s observations on five populations to other population groups. These studies generally confirmed the Dahl relationship, but, to a greater or lesser extent, suffered from a number of uncertainties and potential biases. Often, the data were not derived from one source, but from a variety of studies in the published literature in which unstandardized and often unspecified methods were used, and few data on confounding variables were available. Perhaps the best known of these reports was by Gleibermann, an anthropologist, who examined the relationship between sodium intake and blood pressure across 27 populations. 3 In contrast to Dahl’s reliance on 24 h urinary data to estimate sodium intake, in six of Gleibermann’s populations, the author’s own estimates of sodium intake (6 g salt/day) were used, while in a further ten ‘a quantitative value for mean salt consumption was reported with or without indications as to how it was calculated’. 3

Contributions of Sodium and Chloride to NaCl-Induced Hypertension

The recommendation to avoid high dietary “salt” intake for the prevention and treatment of hypertension is often expressed in terms of dietary sodium. However, a consistent body of evidence suggests that the chloride component of salt is an important contributor to NaCl-induced elevations of blood pressure.1 In several rat models of salt-sensitive hypertension (Dahl salt-sensitive [S] rat, DOCA-salt hypertension, stroke-prone SHR [SHRSP], angiotensin II-induced salt-sensitive hypertension), selective dietary sodium loading, in the absence of chloride, fails to produce hypertension. In various feeding protocols, anions provided with sodium included various combinations of bicarbonate, phosphate, aspartate, glutamate, and glycinate. Overall, the failure of selective dietary sodium loading to produce hypertension in these studies was not related to group differences of body weight, net sodium balance, blood pH, or serum concentrations of sodium, potassium, or chloride. Similarly, a limited number of clinical observations also indicate that blood pressure is not increased in humans by high dietary sodium intakes in the absence of chloride. In 1929, Berghoff et al reported that blood pressure increased in 7 hypertensive individuals on a high NaCl intake, but not on a high sodium bicarbonate intake.2 This observation was subsequently confirmed.3 Similarly, other investigators have also observed that in contrast to the increase of blood pressure induced by a high NaCl intake in hypertensive patients, blood pressure is not increased by a high sodium intake provided as sodium phosphate or sodium citrate.1 Further suggesting a modulating effect of dietary chloride on blood pressure, in hypertensive and normotensive subjects, substitution of dietary NaCl with equimolar sodium bicarbonate leads to a reduction of blood pressure.4,5 Additionally, in hypertensive humans, the reduction of blood pressure by dietary potassium is attenuated by potassium chloride compared with that of potassium citrate.6 Because the blood pressure increment in response …

Oxidative Stress in the Dahl Salt-Sensitive Hypertensive Rat

Oxidative stress has been proposed as important in the pathogenesis of hypertension. Measurement of 8-iso prostaglandin F2α(8-ISO) is introduced for evaluating oxidative stress in vivo. 8-ISO is the major urinary metabolite of F2-isoprostanes and is formed nonenzymatically from the attack of superoxide radicals on arachidonic acid. We examined the oxidative stress level in the Dahl salt-sensitive (Dahl-S) rats and the Dahl salt-resistant (Dahl-R) rats. Dahl-S and Dahl-R rats were fed either a high salt diet (8% NaCl; HS) or low salt diet (0.3% NaCl; LS) for 3 weeks, and systolic blood pressure (SBP) and 24-hr urinary excretion of 8-ISO (U-8-ISO) were measured. In Dahl-S rats, the high salt diet induced hypertension (139 ± 3 mmHg in LS versus 186 ± 2 mmHg in HS, p < .05) and significantly increased the U-8-ISO (24.9 ± 3.6 ng/24 hr in LS versus 63.2 ± 14.6 ng/24 hr in HS, p < .05). No significant difference in blood pressure or U-8-ISO was observed between high-salt and low-salt treated Dahl-R rats. U-8-ISO concentration was correlated with SBP in all four experimental groups (r = 0.866). Moreover, urinary 8-hydroxy-2′-deoxyguanosine (U-8-OHdG), which is one of the most commonly used markers for evaluation of oxidative stress, was higher in Dahl-S-8% rats than in Dahl-S-0.3% rats (136.1 ± 48.4 ng/24 hr in LS versus 322.8 ± 46.7 ng/24 hr in HS, p < .05), and U-8-OHdG was correlated with SBP (r = 0.681) in Dahl-S rats. These results suggest oxygen radicals are involved in the pathogenesis of hypertension.

Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension

Vascular tissues express arginase that metabolizes L-arginine to L-ornithine and urea and thus reduces substrate availability for nitric oxide formation. Dahl salt-sensitive (Dahl-S) rats with salt-induced hypertension show endothelial dysfunction, including decreased vascular nitric oxide formation. This study tests the hypothesis that increased vascular arginase activity contributes to endothelial dysfunction in hypertensive Dahl-S rats. Male Dahl-S rats (5-6 wk) were placed on high (8%) or low (0.3%) NaCl diets for 4 wk. With respect to the low-salt group, mean arterial blood pressure was increased in the high-salt animals. Immunohistochemical stainings for arginase I and II were enhanced in arterioles isolated from high-salt Dahl-S rats. Experiments used isolated Krebs buffer-superfused first-order gracilis muscle arterioles with constant pressure (80 mmHg) and no luminal flow or constant midpoint but altered endpoint pressures to establish graded levels of luminal flow (0-50 microl/min). In high-salt arterioles, responses to an endothelium-dependent vasodilator acetylcholine (1 nmol/l to 3 micromol/l) and flow-induced dilation were decreased. Acute in vitro treatment with an inhibitor of arginase, 100 micromol/l (S)-(2-boronoethyl)-L-cystine, or the nitric oxide precursor, 1 mmol/l L-arginine, similarly enhanced acetylcholine and flow-induced maximal dilations and abolished the differences between high- and low-salt arterioles. These data show that arteriolar arginase expression is increased and that endothelium-dependent vasodilation is decreased in high-salt Dahl-S rats. Acute pretreatment with an arginase inhibitor or with L-arginine restores endothelium-dependent vasodilation and abolishes the differences between high- and low-salt groups. These results suggest that enhanced vascular arginase activity contributes to endothelial dysfunction in Dahl-S rats with salt-induced hypertension and identifies arginase as a potential therapeutic target to prevent endothelial dysfunction.

Heme oxygenase-derived carbon monoxide promotes arteriolar endothelial dysfunction and contributes to salt-induced hypertension in Dahl salt-sensitive rats

Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. After 4 wk of high-salt diet, Dahl salt-sensitive (Dahl-S) rats display hypertension, increased vascular HO-1 expression, and attenuated vasodilator responses to ACh that can be completely restored by acute treatment with an inhibitor of HO. In this study, we examined the temporal development of HO-mediated endothelial dysfunction in isolated pressurized first-order gracilis muscle arterioles, identified the HO product responsible, and studied the blood pressure effects of HO inhibition in Dahl-S rats on a high-salt diet. Male Dahl-S rats (5-6 wk) were placed on high-salt (8% NaCl) or low-salt (0.3% NaCl) diets for 0-4 wk. Blood pressure increased gradually, and responses to an endothelium-dependent vasodilator, ACh, decreased gradually with the length of high-salt diet. Flow-induced dilation was abolished in hypertensive Dahl-S rats. Acute in vitro pretreatment with an inhibitor of HO, chromium mesoporphyrin (CrMP), restored endothelium-dependent vasodilation and abolished the differences between groups. The HO product CO prevented the restoration of endothelium-dependent dilation by CrMP. Furthermore, administration of an HO inhibitor lowered blood pressure in Dahl-S rats with salt-induced hypertension but did not do so in low-salt control rats. These results suggest that hypertension and HO-mediated endothelial dysfunction develop gradually and simultaneously in Dahl-S rats on high-salt diets. They also suggest that HO-derived CO underlies the impaired endothelial dysfunction and contributes to hypertension in Dahl-S rats on high-salt diets.

Genome-Wide Scan Identifies Novel QTLs for Cholesterol and LDL Levels in F2[Dahl R×S]-Intercross Rats

Hypercholesterolemia is a significant risk factor for coronary artery disease development. Genes influencing nonmonogenic hypercholesterolemia susceptibility in humans remain to be identified. Animal models are key investigative systems because major confounding variables such as diet, activity, and genetic background can be controlled. We performed a 121-marker, total genome-analysis of an F2[Dahl RxS]-intercross selected for contrasting parental strain susceptibilities for hyperlipidemia on regular rat diets at 6 months of age. Quantitative traits studied were plasma total cholesterol, triglyceride, HDL, and LDL levels adjusted for obesity. Genome-wide analysis of 200 F2-intercross male rats detects two QTLs with highly significant linkage for total cholesterol (TC) on chromosome (chr) 5-133.3 Mbp (LOD 5.8), and chr5-54.2 Mbp (LOD 4.8), and two QTLs with significant linkage for TC: on chromosome 8, chr8-60.4 Mbp (LOD 3.8), and chromosome 2, chr2-243.5 Mbp (LOD 3.4). A QTL for LDL with significant linkage is detected on chromosome 5, chr5-104 Mbp (LOD 3.7). These QTLs contribute from 7% to 12% of total trait variance, respectively, with Dahl-S allele effects resulting in increased TC and LDL levels consistent with hyperlipidemia susceptibility in the parental Dahl-S rat strain. Predicted QTL-peaks do not coincide with previous genome scans. Human homologues of two TC-QTLs span genes listed in a LocusLink profile for cholesterol. Only suggestive loci were detected for HDL and total triglyceride levels. Altogether, the data demonstrates the contribution of multiple QTLs to hypercholesterolemia making a multipathway pathogenic framework imperative. QTL-peak candidate genes delineated are syntenic between rat and human genomes, increasing clinical relevance and mandating further study.

Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo

Thiazolidinediones have been shown to activate AMP-activated protein kinase activity in cultured cells. Whether they have a similar effect in vivo and if so whether it is physiologically relevant is not known. To assess these questions, we examined the effects of pioglitazone, administered orally to intact rats, on AMPK phosphorylation (AMPK-P) (a measure of its activation) and acetyl CoA carboxylase (ACC) activity and malonyl CoA concentration in rat liver and adipose tissue. In the first study, measurements were made in the Dahl-salt-sensitive rat (Dahl-S), a strain of Sprague–Dawley rat with endogenous hypertriglyceridemia and high levels of malonyl CoA that are restored to control values by pioglitazone. Treatment with pioglitazone (20 mg/kg bw/day for 3 weeks) did not significantly increase either P-AMPK or P-ACC (which varies inversely with ACC activity) in control rats. However, in the Dahl-S rats values for AMPK-P and ACC-P were 50% lower than in control rats and were doubled by pioglitazone treatment. In a second study, the effects of two weeks treatment with pioglitazone (3 mg/kg bw/day administered orally) were evaluated in Wistar rats. Under basal conditions (no manipulation of the animals), pioglitazone increased AMPK phosphorylation by twofold and decreased ACC activity and the concentration of malonyl CoA by 50% in liver. Following a euglycemic–hyperinsulinemic clamp (6 h), 50% decreases in AMPK and ACC phosphorylation (indicating an increase in its activity) and comparable increases in malonyl CoA concentration were observed in liver and adipose tissue. In both tissues, pre-treatment with pioglitazone prevented these changes. Where studied (in Wistar rats under basal conditions) treatment with pioglitazone decreased the concentration of ATP by 1/3 and increased the concentration of ADP and AMP in liver. The results indicate that treatment with pioglitazone can increase AMPK activity in rat liver and adipose tissue in a variety of circumstances. They also suggest that this activation of AMPK may be mediated by a change in cellular energy state. Whether these effects of pioglitazone contribute to its insulin-sensitizing and other actions in vivo remains to be determined.

Pyridoxine 5 ′-phosphate oxidase is a candidate gene responsible for hypertension in Dahl-S rats

To identify candidate genes responsible for hypertension in Dahl salt-sensitive rats (Dahl-S), an oligonucleotide microarray analysis was performed to find differentially expressed genes in kidneys of Dahl-S and Lewis rats. We obtained 101 F2 male rats from Dahl-S and Lewis rats and performed precise measurements of blood pressure (BP) and heart rate by telemetric monitoring at 14 weeks of age after 9 weeks of salt-loading. The correlation analysis between genotypes of differentially expressed genes and BP in F2 rats indicated that pyridoxine 5 ′-phosphate oxidase ( Pnpo) and catecholamine- O-methyltransferease ( Comt) showed a highly significant association with BP. However, in the case of Comt, the Dahl-S genotype correlated with low BP. Short/branched chain acyl-CoA dehydrogenase and Sah also showed a significant association with systolic blood pressure. The present study provided evidence that Pnpo is a candidate gene responsible for hypertension in Dahl-S rats.

High sodium intake stimulates endogenous sodium pump inhibitor, marinobufagenin, and increases blood pressure in pregnant rats

Previous studies have implicated endogenous digitalis-like sodium pump ligands in pathogenesis of preeclampsia. Recently, we have shown that an endogenous bufadienolide, marinobufagenin (MBG), an inhibitor of ouabain resistant alpha-1 Na/K-ATPase, rather than endogenous ouabain (EO) becomes elevated and contributes to hypertension in patients with preeclampsia (Lopatin et al, J Hypertens 1999; 17: 1179-87) and in Dahl-S rats on a high NaCl intake (Fedorova et al, Circulation 2002; 105: 1122-27). We hypothesized that in pregnant rats on a high NaCl intake MBG increases and contributes to blood pressure (BP) elevation. Renal excretion of MBG, EO and sodium, systolic BP, and plasma renin activity (PRA) were compared in non-pregnant and pregnant Sprague-Dawley rats (225-250 g) during last week of pregnancy. Pregnant animal consumed either water or NaCl solution, 0.9% or 1.8%. As compared with non-pregnant rats, last week of pregnancy was associated with decreased systolic BP, increased PRA and moderate increases in renal excretion of MBG and EO. NaCl supplementation induced graded increases in renal Na excretion, in systolic BP and renal excretion of MBG and with concomitant decrease in PRA (Table). Supplementation with 1.8% NaCl was also associated with fetal growth retardation. Our results demonstrate that in rats NaCl supplementation during the last week of pregnancy induces preeclampsia-like symptoms including elevation of BP, proteinuria, suppression of PRA and fetal growth retardation. Elevations of BP are associated with enhanced excretion of MBG, which makes MBG a potential target for therapy. One-way ANOVA followed by Newman-Keuls test. -P<0.05, -P<0.01 vs. nonpregnant rats; -P<0.05, -P<0.01 vs. pregnant rats without NaCl supplementation. n=12–15 for each group. One-way ANOVA followed by Newman-Keuls test. -P<0.05, -P<0.01 vs. nonpregnant rats; -P<0.05, -P<0.01 vs. pregnant rats without NaCl supplementation. n=12–15 for each group.

Cross talk between central and peripheral digitalis-like sodium pump ligands (SPL) in Dahl-S rats (DS) following NaCl loading

Cross talk between central and peripheral digitalis-like sodium pump ligands (SPL) in Dahl-S rats (DS) following NaCl loading

Myocardial PKC beta2 and the sensitivity of Na/K-ATPase to marinobufagenin are reduced by cicletanine in Dahl hypertension.

Marinobufagenin (MBG), an endogenous ligand of alpha-1 Na/K-ATPase, becomes elevated and contributes to hypertension in NaCl-loaded Dahl-S rats (DS). Protein kinase C (PKC) phosphorylates alpha-1 Na/K-ATPase and increases its MBG sensitivity. Cicletanine, an antihypertensive compound with PKC-inhibitory activity, reverses MBG-induced Na/K-ATPase inhibition and vasoconstriction. We hypothesized that increased PKC levels in sodium-loaded hypertensive DS would sensitize alpha-1 Na/K-ATPase to MBG and that PKC inhibition by cicletanine would produce an opposite effect. We studied the effects of cicletanine on systolic blood pressure, left ventricular PKC isoforms, cardiac alpha-1 Na/K-ATPase levels, and sensitivity to MBG in hypertensive DS. Seven DS received 50 mg x kg(-1) x d(-1) cicletanine, and 7 DS received vehicle during 4 weeks of an 8% NaCl diet. Vehicle-treated rats exhibited an increase in blood pressure, left ventricular mass, MBG excretion (74+/-11 vs 9+/-1 pmol/24 h, P<0.01), myocardial alpha-1 Na/K-ATPase protein, and PKC beta2 and delta. The sensitivity of Na/K-ATPase to MBG was enhanced at the level of high-affinity binding sites (IC50, 0.8 vs 4.4 nmol/L, P<0.01). Cicletanine-treated rats exhibited a 56-mm Hg reduction in blood pressure (P<0.01) and a 30% reduction in left ventricular weight, whereas cardiac alpha-1 Na/K-ATPase protein and MBG levels were unchanged. In cicletanine-treated rats, PKC beta2 was not increased, the sensitivity of Na/K-ATPase to MBG was decreased (IC50=20 micromol/L), and phorbol diacetate-induced alpha-1 Na/K-ATPase phosphorylation was reduced versus vehicle-treated rats. In vitro cicletanine treatment of sarcolemma from vehicle-treated rats also desensitized Na/K-ATPase to MBG, indicating that this effect was not solely attributable to a reduction in blood pressure. Thus, PKC-induced phosphorylation of cardiac alpha-1 Na/K-ATPase is a likely target for cicletanine treatment.

Effects of nicotine and dietary salt on a learned blood pressure response in Dahl-S rats.

We examined the effects of chronic nicotine exposure and dietary salt on the arterial blood pressure (BP) changes learned in response to an acute behavioral stress in the Dahl salt-sensitive rat. Four groups were tested: low salt + vehicle; low salt + nicotine; high salt + vehicle; and high salt + nicotine. Rats were fed a low-salt (0.08% NaCl) or a high-salt (8% NaCl) diet for 4 wk; 2.4 mg. kg(-1). day(-1) nicotine or vehicle was given via an implanted osmotic minipump for the last 2 wk. All rats were conditioned by following one tone (CS+) with a 0.5-s tail shock; another tone (CS-) was never followed by shock. CS+ in low salt + vehicle and high salt + vehicle-treated rats evoked an initial arterial BP increase (C(1)), a component of the startle response, and an ensuing, smaller, but more sustained, pressor response (C(2)), which is acquired with training. In these rats, both C(1) and C(2) evoked by CS- were significantly smaller than those to CS+, demonstrating that these groups discriminated between the two tests. Conversely, although the low salt + nicotine-treated rats had both the C(1) and C(2) components of the conditional arterial pressure response, they did not discriminate between CS+ and CS-. Finally, the high salt + nicotine group failed to both discriminate between tones and acquire (i.e., learn) the C(2) response. The unconditional response to shock did not differ between groups. We conclude that combined exposure to high salt and to nicotine inhibits the salt-sensitive animal's acquisition of a learned conditional BP response, perhaps because nicotine acts preferentially on those central processes required for associative learning versus those involved in orientating to external stimuli.

Treatment of hypertension with oral taurine: experimental and clinical studies.

Oral taurine treatment has been studied extensively as a hypotensive agent. Several rat models of hypertension have been used to prove that dietary taurine supplementation can alleviate high blood pressure, among other cardiovascular problems. Experimental models mentioned in this review are the spontaneously hypertensive rat, the DOCA-salt rat, the Dahl-S rat, the renovascular hypertensive rat, the hyperinsulinemic rat and the ethanol-treated rat. The beneficial effects of taurine were also demonstrated in studies involving human subjects suffering essential hypertension. Taurine supplementation of 6 g/day for as little as 7 days resulted in measurable decreases in blood pressure in these patients. In both rat and human studies, the effects of taurine appeared to be dependent on the modulation of an overactive sympathetic system. However, taurine has positive effects on other types of cardiovascular problems and thus may act through more than one mechanism.