High-salt Diet Rats Research Articles

High salt diet accelerates skin aging in wistar rats: an 8-week investigation of cell cycle inhibitors, SASP markers, and oxidative stress.

Recent studies have shown that the high salt diet (HSD) is linked to increased dermal pro-inflammatory status and reduced extracellular matrix (ECM) expression in inflamed skin of mice. Decreased ECM content is a known aging phenotype of the skin, and alterations in ECM composition and organization significantly contribute to skin aging. This study aimed to determine whether a high salt diet accelerates skin aging and to identify the time point at which this effect becomes apparent. Wistar rats were randomly divided into normal diet and high salt diet groups and fed continuously for 8weeks. Skin samples were collected at weeks 7 and week 8. Skin pathological sections were evaluated and levels of cell cycle inhibitors, senescence-associated secretory phenotype (SASP), oxidative stress and vascular regulatory factors (VRFs) were examined. Correlation analyses were performed to reveal the effect of a high salt diet as an extrinsic factor on skin aging and to analyse the correlation between a high salt diet and intrinsic aging and blood flow status. At week 8, HSD rats exhibited thickened epidermis, thinned dermis, and atrophied hair follicles. The expression of cell cycle inhibitors and oxidative stress levels were significantly elevated in the skin of HSD rats at both week 7 and week 8. At week 7, some SASPs, including TGF-β and PAI-1, were elevated, but others (IL-1, IL-6, IL-8, NO) were not significantly changed. By week 8, inflammatory molecules (IL-1, IL-6, TGF-β), chemokines (IL-8), proteases (PAI-1), and non-protein molecules (NO) were significantly increased. Notably, despite elevated PAI-1 levels suggesting possible blood hypercoagulation, the ET-1/NO ratio was reduced in the HSD group at week 8. The data suggest that a high salt diet causes skin aging by week 8. The effect of a high salt diet on skin aging is related to the level of oxidative stress and the expression of cell cycle inhibitors. Additionally, a potential protective mechanism may be at play, as evidenced by the reduced ET-1/NO ratio, which could help counteract the hypercoagulable state and support nutrient delivery to aging skin.

Deleterious effect in endothelin receptor–mediated coronary artery smooth muscle contractility in high-salt diet rats

Background and aimsHigh-salt diet has been suggested to increase the risk of heart disease. However, the mechanisms underlying coronary artery tension dysfunction caused by high-salt diet are unclear. Previous studies have shown that coronary artery spasm is often induced by endothelin-1 (ET-1) and thromboxane, leading to myocardial ischemia, while the store-operated Ca2+ entry (SOCE) function of coronary smooth muscle is very important in this process. Methods and resultsTension measurements of endothelium-denuded coronary artery ring segments showed that vasocontraction induced by U46619, ET-1, orSTIM1/Orai1-mediated SOCE was significantly lower in 4% high-salt diet rats than in control rats fed a regular diet. The results of western blotting and immunohistochemistry assays showed lower expression levels of endothelial receptors ETA and ETB, STIM1 and Orai1 in coronary artery of high-salt intake rats compared with control rats. Fibrosis was observed by using Masson's trichrome staining and picrosirius red staining. The plasma ET-1 concentration in high-salt diet rats was significantly higher than that of controls. The interventricular septum and posterior wall of high-salt diet rats were significantly thickened. ConclusionOur findings indicated that coronary artery tension was significantly decreased in 4% high-salt diet rats and that this decrease may be due to the change of endothelin receptor and its downstream pathway SOCE related protein expression in coronary artery. Coronary fibrosis was observed in rats fed with high-salt diet. This study provides potential mechanistic insights into high-salt intake–induced heart disease.

Genistein Alleviates Oxidative Stress and Inflammation in the Hypothalamic Paraventricular Nucleus by Activating the Sirt1/Nrf2 Pathway in High Salt-Induced Hypertension.

Hypertension caused by a high-salt (HS) diet is one of the major causes of cardiovascular diseases. Underlining pathology includes oxidative stress and inflammation in the hypothalamic paraventricular nucleus (PVN). This study investigates genistein's (Gen) role in HS-induced hypertension and the underlying molecular mechanism. We placed male Wistar rats on HS (8% NaCl) or normal salt diet (0.3% NaCl). Then, we injected bilateral PVN in rats with Gen, vehicle, or nicotinamide (NAM) for 4weeks. Tail cuff was used weekly to assess the systolic pressure, diastolic pressure, and mean arterial pressure (MAP). Cardiac hypertrophy was analyzed by heart weight/body weight ratio and wheat germ agglutinin staining. ELISA kits, Western blot, or dihydroethidium staining determined the levels of inflammatory cytokines and oxidative stress markers. Western blot measured protein levels of Sirt1, Ac-FOXO1, Nrf2, NQO-1, HO-1, and gp91phox. Our result showed that PVN infusion of Gen significantly reduced the increase of systolic pressure, diastolic pressure, and MAP induced by an HS diet. Additionally, there was a decrease in cardiac hypertrophy and the levels of inflammatory cytokines in PVN and plasma. Meanwhile, PVN infusion of Gen notably inhibited the levels of oxidized glutathione and superoxide dismutase and improved the glutathione level and total antioxidant capacities and superoxide dismutase activities. It also decreased the level of reactive oxygen species and gp91phox expression in PVN. Furthermore, Gen infusion markedly increases the Sirt1, Nrf2, HO-1, and NQO-1 levels and decreases the Ac-FOXO1 level. However, PVN infusion of NAM could significantly block these changes induced by Gen in HS diet rats. Our results demonstrated that PVN infusion of Gen could inhibit the progression of hypertension induced by an HS diet by activating the Sirt1/Nrf2 pathway.

Nitric Oxide Alleviated High Salt-Induced Cardiomyocyte Apoptosis and Autophagy Independent of Blood Pressure in Rats.

The present study aimed to explore whether high-salt diet (HSD) could cause cardiac damage independent of blood pressure, and whether nitric oxide (NO) could alleviate high-salt–induced cardiomyocyte apoptosis and autophagy in rats. The rats received 8% HSD in vivo. H9C2 cells or primary neonatal rat cardiomyocytes (NRCM) were treated with sodium chloride (NaCl) in vitro. The levels of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2, LC3 II/LC3 I, Beclin-1 and autophagy related 7 (ATG7) were increased in the heart of HSD rats with hypertension (HTN), and in hypertension-prone (HP) and hypertension-resistant (HR) rats. Middle and high doses (50 and 100 mM) of NaCl increased the level of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2, LC3 II/LC3 I, Beclin-1, and ATG7 in H9C2 cells and NRCM. The endothelial NO synthase (eNOS) level was increased, but p-eNOS level was reduced in the heart of HSD rats and H9C2 cells treated with 100 mM NaCl. The level of NO was reduced in the serum and heart of HSD rats. NO donor sodium nitroprusside (SNP) reversed the increases of cleaved-caspase 3/caspase 3, cleaved-caspase 8/caspase 8, Bax/Bcl2 induced by NaCl (100 mM) in H9C2 cells and NRCM. SNP treatment attenuated the increases of cleaved-caspase 3/caspase 3, Bax/Bcl2, LC3 II/LC3 I, Beclin-1, and ATG7 in the heart, but had no effect on the blood pressure of HSD rats with HR. These results demonstrated that HSD enhanced cardiac damage independently of blood pressure. Exogenous NO supplementarity could alleviate the high salt–induced apoptosis and autophagy in cardiomyocytes.

Chromosome 2 Fragment Substitutions in Dahl Salt-Sensitive Rats and RNA Sequencing Identified Enpep and Hs2st1 as Vascular Inflammatory Modulators.

Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2BN/Mcwi; consomic S2B) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[rs13453786-rs66377062]/Aek; S2Ba) and middle segment (SS.BN-[rs106982173-rs65057186]/Aek; S2Bb) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2Ba rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase (Enpep) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 (Hs2st1) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1, associated with increased vascular inflammation under HSD.

142-LB: Very Low Dose SGLT2 Inhibition Is as Effective as Angiotensin II Receptor Blockage for Cardiac and Renal Fibrosis in High Salt 5/6 Nephrectomized Rats

SGLT2 inhibition has beneficial effects on cardiovascular and renal endpoints but the lowest effective dose is unknown. Effects of the SGLT2 inhibitor, empagliflozin (EMPA) were evaluated in nondiabetic, 5/6 nephrectomy (5/6 Nx) rats. Rats were treated for 8 weeks: Sham + normal diet + placebo (PBO); 5/6Nx + 2% high salt diet (HSD) + PBO; 5/6Nx + HSD + EMPA 1.2 mg/kg/day); 5/6Nx + HSD + telmisartan (5 mg/kg/day); 5/6Nx + HSD + linagliptin (3 mg/kg/day). EMPA increased urinary glucose excretion at dosages eight times lower than currently used low doses. 5/6Nx HSD rats treated with EMPA showed significantly (-29.95/-25.00 mmHg, p<0.001) lower blood pressure (BP) and significantly reduced renal (-33.67%; p<0.05) and cardiac (- 34.85%; p<0.05) fibrosis vs. PBO. Effects of EMPA on BP and renal and cardiac fibrosis were comparable to telmisartan (BP: -31.89/-24.24 mmHg, p<0.001; renal fibrosis -43.96%; p<0.01; cardiac fibrosis: -36.37%; p<0.01) and effects on fibrosis were comparable to linagliptin (renal fibrosis -41.54%; p<0.05; cardiac fibrosis: -50.01%; p<0.001). Many genes were differentially regulated among groups, e.g., transferrin receptor (Trfc), A-kinase anchor protein 1 (Akap1) and mitogen-activated protein kinase 9 (Mapk9) were affected by EMPA and telmisartan while Caspase 1 (Casp1) gene expression was altered by EMPA only in the heart. In the kidney, both EMPA and linagliptin led to differential regulation of pleckstrin homology domain-containing family A member 1 (Plekha1), while only EMPA resulted in differential expression of prostaglandin E synthase (Ptges) and G protein-coupled receptor, class C, group 5, member A (Gprc5a) gene expression. Very low dose EMPA was almost as effective as standard dosages of telmisartan or linagliptin in counteracting BP increase and cardiac and renal fibrosis. The underlying mechanism involves regulation of genes involved in cardiac and renal fibrosis. Disclosure S. Zeng: None. A.A. Hasan: Research Support; Self; Boehringer Ingelheim International GmbH. D. Delic: None. Y. Xiong: None. C. Chu: None. L. Yin: None. S. Frankenreiter: Employee; Self; Boehringer Ingelheim Pharma GmbH & Co. KG. Other Relationship; Self; Evotec International GmbH. B. Krämer: Advisory Panel; Self; Boehringer Ingelheim International GmbH. T. Klein: Employee; Self; Boehringer Ingelheim International GmbH. B. Hocher: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Funding Boehringer Ingelheim & Eli Lilly and Company Diabetes Alliance

A dipeptidyl peptidase-IV inhibitor improves diastolic dysfunction in Dahl salt-sensitive rats

To date, there is no established treatment for heart failure with preserved ejection fraction (HFpEF). Dipeptidyl peptidase-IV (DPP-IV) inhibitors reportedly have improved not only diabetes mellitus but also heart failure with systolic dysfunction in experimental models. We investigated the effects of a DPP-IV inhibitor on HFpEF in rats. Dahl salt-sensitive rats were fed either high-salt (high-salt diet (HSD): 8% NaCl) or low-salt diets (0.3% NaCl) from 6.5 weeks of age. They were then treated with or without a DPP-IV inhibitor, vildagliptin (10 mg/kg/day, orally), from 11 weeks of age for 9 weeks and analyzed at the age of 20 weeks. HSD rats mimicked the pathophysiology of HFpEF. There were no differences in heart rate, blood pressure, left ventricular (LV) systolic function, or the extent of LV hypertrophy between HSD rats with or without vildagliptin. However, vildagliptin decreased LV end-diastolic pressure, the most reliable hemodynamic parameter of HFpEF in HSD rats. Vildagliptin also decreased the LV distensibility index, a sensitive marker of LV diastolic function in HSD rats. Vildagliptin decreased the expression of collagen genes in HSD hearts and attenuated LV interstitial fibrosis (HSD with vehicle and vildagliptin, 2.9% vs. 1.9%; P < 0.05). Furthermore, vildagliptin administration reduced both plasma renin activity and aldosterone concentrations in HSD rats. A DPP-IV inhibitor, vildagliptin, improved the severity of LV fibrosis, and thus, diastolic dysfunction of HFpEF in Dahl salt-sensitive hypertensive rats. DPP-IV inhibitors are promising medicines for treatment of HFpEF in patients with diabetes mellitus.

Exercise Training Attenuates Proinflammatory Cytokines, Oxidative Stress and Modulates Neurotransmitters in the Rostral Ventrolateral Medulla of Salt-Induced Hypertensive Rats

Background/Aims: Exercise training (ExT) was associated with cardiovascular diseases including hypertension. The rostral ventrolateral medulla (RVLM) is a key region for central control of blood pressure and sympathetic nerve activity. Therefore, this study aimed to investigate the mechanisms within RVLM that can influence exercise training induced effects in salt-induced hypertension. Methods: Male Wistar rats were fed with a normal salt (0.3%) (NS) or a high salt (8%) (HS) diet for 12 weeks to induce hypertension. Then these rats were given moderate-intensity ExT for a period of 12 weeks. RVLM was used to determine glutamate and gamma-aminobutyric acid (HPLC), phosphorylated IKKβ, Fra-LI, 67-kDa isoform of glutamate decarboxylase (GAD67), proinflammatory cytokines (PIC) and NADPH-oxidase (NOX) subunits expression (Immunohistochemistry and Immunofluorescence, Western blotting). PIC and NF-κB p65 activity in the plasma were evaluated by ELISA studies. Renal sympathetic nerve activity (RSNA) was recorded and analyzed using the PowerLab system. Results: High salt diet resulted in increased mean arterial pressure and cardiac hypertrophy. These high salt diet rats had higher RVLM levels of glutamate, PIC, phosphorylated IKKβ, NF-κB p65 activity, Fra-LI, superoxide, NOX-2 (gp91phox) and 4, and lower RVLM levels of gamma-aminobutyric acid and GAD67, and higher plasma levels of PIC, norepinephrine, and higher RSNA. ExT attenuated these changes in salt-induced hypertensive rats. Conclusions: These findings suggest that high salt diet increases the activity of NF-κB and the levels of PIC and oxidative stress, and induces an imbalance between excitatory and inhibitory neurotransmitters in the RVLM. ExT attenuates hypertension and cardiac hypertrophy partially mediated by attenuating oxidative stress and modulating neurotransmitters in the RVLM.

OP.1C.01] HIGH SALT DIET INCREASES AORTIC STIFFNESS AND PRESSURE PULSE AMPLIFICATION IN RAT

Objective: High salt consumption is associated with increased blood pressure and vascular changes in humans. This association is confounded by other lifestyle factors that could equally influence cardiovascular function. This study examines effects of high salt diet on aortic function in a controlled animal study. Design and method: Sprague-Dawley rats were fed either high salt (8% sodium chloride, n = 6) or normal chow (0.26% sodium chloride, n = 5) from weaning. Body weight (BW), nose-to-rump length, tail-cuff systolic blood pressure (SBP), water and food intake, and urine output were measured. At 14 to 17 weeks, rats were anaesthetised and thoracic and abdominal aortic pressure (invasive solid-state catheters) recorded over a physiological mean arterial pressure (MAP) range (60 to 150 mmHg, induced via intra-venous infusion of phenylephrine and sodium nitroprusside, 30 μg/kg/min). Aortic pulse wave velocity (aPWV) and thoracic to abdominal aortic pulse pressure amplification (PPA) were calculated. Left ventricular (LV) and kidney mass were recorded post-mortem. Results Results: High salt diet rats had lower BW (average across all age: 451 ± 57 g, control 498 ± 68 g, p = 0.03), body mass index (0.79 ± 0.08 g/cm2, control 0.82 ± 0.10 g/cm2, p < 0.001) and kidney mass (kidney/BW 0.50 ± 0.09%, control 0.39 ± 0.05%, p = 0.01) without LV hypertrophy (LV/BW 0.24 ± 0.04%, control 0.22 ± 0.03%, p = 0.16). Food intake was similar (high salt 20 ± 3 g/day, control 19 ± 5 g/day, p = 0.36). High salt diet rats drank more (65 ± 10 ml/day, control 22 ± 6 ml/day, p < 0.001) with correspondingly higher urine output (55 ± 9 ml/day, control 15 ± 6 ml/day p < 0.001); conscious SBP was greater (125 ± 12 mmHg, control 117 ± 13 mmHg, p < 0.05). High salt diet resulted in higher aPWV (high salt aPWV across MAP range 60 to 150 mmHg, 4.3 ± 0.2 to 5.4 ± 0.2 m/s, control 3.6 ± 0.3 to 3.9 ± 1.1 m/s, p < 0.001 at each 5 mmHg MAP interval). PPA was greater in high salt diet rats (high salt PPA across MAP range 60 to 150 mmHg, 0.78 ± 0.04 to 1.53 ± 0.27, control 0.62 ± 0.14 to 1.25 ± 0.17, p < 0.001 at each 5 mmHg MAP interval). Conclusions: A high salt diet induced moderately higher arterial blood pressure, greater aortic stiffness and higher PPA, indicating marked changes in transmission characteristics of the aorta, including altered stiffness gradient across the aorta and changed peripheral wave reflection characteristics.

IMCD to commercialize DuPont's enzymes in Europe and North Africa

Nephron specific regulation of chloride channel CLC-K2 mRNA in the ratThis study investigated the influence of salt intake on the nephron specific gene expression of the kidney chloride channel CLC-K2. To this end, male Sprague-Dawley rats were fed a low (0.02% wt/wt), normal (0.6% wt/wt), or high salt (8% wt/wt) diet for ten days, or they received the loop diuretic furosemide (12 mg/kg/day) for six days.Expression and regulation of messenger RNA for CLC-K2 Was demonstrated by RNase protection assay and in situ hybridization in kidney cortex, outer medulla and inner medulla. Tubular localization and regulation were determined precisely by reverse transcription-polymerase chain reaction (RT-PCR) and real time PCR of microdissected nephron segments.In situ hybridization analysis and RNase protection assay of the total kidney revealed a down-regulation of CLC-K2 mRNA in the high salt diet rats and an up-regulation of CLC-K2 mRNA in furosemide treated rats, which were restricted to the outer medulla. Microdissection of collagenase treated kidney revealed CLC-K2 mRNA expression in the outer medullary thick ascending limb (mTAL), cortical thick ascending limb (cTAL), distal convoluted tubule (DCT), connecting tubule and cortical collecting duct (CNT/CCD), and outer medullary collecting duct (OMCD), whereas no signals were detected in proximal convoluted and straight tubules (PCT and PST), descending thin limb from the outer medulla (dTL), descending and ascending thin limb from the inner medulla (TL), inner medullary collecting duct (IMCD) and glomeruli (glom). Using RT-PCR and real time PCR, the changing levels of CLC-K2 mRNA after furosemide treatment or high salt diet were restricted to the mTAL, whereas CLC-K2 mRNA levels in cTAL and OMCD were not changed in furosemide or high salt rats compared to time paired controls.Given that CLC-K2 expressed in the thick ascending limb of Henle's loop is responsible for net chloride reabsorption in this part of the nephron, our findings suggest that in states of surplus salt and in states of severe salt deprivation, selective regulation of CLC-K2 mRNA plays a role in the adaptation of the kidney to different salt loads.

The accumulation of brain water-free sodium is associated with ischemic damage independent of the blood pressure in female rats

Estrogen deficiency worsens ischemic stroke outcomes. In ovariectomized (OVX+) rats fed a high-salt diet (HSD), an increase in the body Na+/water ratio, which characterizes water-free Na+ accumulation, was associated with detrimental vascular effects independent of the blood pressure (BP). We hypothesized that an increase in brain water-free Na+ accumulation is associated with ischemic brain damage in OVX+/HSD rats. To test our hypothesis we divided female Wistar rats into 4 groups, OVX+ and OVX− rats fed HSD or a normal diet (ND), and subjected them to transient cerebral ischemia. The brain Na+/water ratio was increased even in OVX+/ND rats and augmented in OVX+/HSD rats. The increase in the brain Na+/water ratio was positively correlated with expansion of the cortical infarct volume without affecting the BP. Interestingly, OVX+ was associated with the decreased expression of ATP1α3, a subtype of the Na+ efflux pump. HSD increased the expression of brain Na+ influx-related molecules and the mineralocorticoid receptor (MR). The pretreatment of OVX+/HSD rats with the MR antagonist eplerenone reduced brain water-free Na+ accumulation, up-regulated ATP1α3, down-regulated MR, and reduced the cortical infarct volume. Our findings show that the increase in the brain Na+/water ratio elicited by estrogen deficiency or HSD is associated with ischemic brain damage BP-independently, suggesting the importance of regulating the accumulation of brain water-free Na+. The up-regulation of ATP1α3 and the down-regulation of MR may provide a promising therapeutic strategy to attenuate ischemic brain damage in postmenopausal women.

Transcription factor avian erythroblastosis virus E26 oncogen homolog-1 is a novel mediator of renal injury in salt-sensitive hypertension.

Transcription factor E26 transformation-specific sequence-1 (ETS-1) is a transcription factor that regulates the expression of a variety of genes, including growth factors, chemokines, and adhesion molecules. We recently demonstrated that angiotensin II increases the glomerular expression of ETS-1 and that blockade of ETS-1 ameliorates the profibrotic and proinflammatory effects of angiotensin II. The Dahl salt-sensitive rat is a paradigm of salt-sensitive hypertension associated with local activation of the renin-angiotensin system. In these studies, we determined whether: (1) salt-sensitive hypertension is associated with renal expression of ETS-1 and (2) ETS-1 participates in the development of end-organ injury in salt-sensitive hypertension. Dahl salt-sensitive rats were fed a normal-salt diet (0.5% NaCl diet) or a high-salt diet (4% NaCl) for 4 weeks. Separate groups on high-salt diet received an ETS-1 dominant-negative peptide (10 mg/kg/d), an inactive ETS-1 mutant peptide (10 mg/kg/d), the angiotensin II type 1 receptor blocker candesartan (10 mg/kg/d), or the combination high-salt diet/dominant-negative peptide/angiotensin II type 1 receptor blocker for 4 weeks. High-salt diet rats had a significant increase in the glomerular expression of the phosphorylated ETS-1 that was prevented by angiotensin II type 1 receptor blocker. ETS-1 blockade reduced proteinuria, glomerular injury score, fibronectin expression, urinary transforming growth factor-β excretion, and macrophage infiltration. Angiotensin II type 1 receptor blocker reduced proteinuria, glomerular injury score, and macrophage infiltration, whereas concomitant ETS-1 blockade and angiotensin II type 1 receptor blocker had additive effects and reduced interstitial fibrosis. Our studies demonstrated that salt-sensitive hypertension results in increased glomerular expression of phosphorylated ETS-1 and suggested that ETS-1 plays an important role in the pathogenesis of end-organ injury in salt-sensitive hypertension.

A high salt diet alters pressure-induced mechanical activity of the rat lymphatics with enhancement of myogenic characteristics.

The lymphatic system has become a new player for pathogenesis in salt-sensitive hypertension animals. A high salt diet (HSD) evokes accumulation of Na(+) in the skin of rodents. In response to increase in Na(+)-proteoglycan complex, infiltrated macrophages stimulate secretion of vascular endothelial growth factor (VEGF)-C. Macrophage-derived VEGF-C increases density of the dermal lymph capillaries, indicating that lymphangiogenesis is advantageous to hypertensive animals by buffering elevated blood pressure. However, the effects of a high salt diet (HSD) on changes in mechanical activity of collecting lymph vessels, which directly connect with lymph capillaries, have not yet been determined. Changes in mechanical activity of isolated collecting lymphatics in normal salt diet (NSD) and HSD rats in response to increase in intraluminal pressures were measured by video-microscopy. HSD vessels had smaller % active diameters (maximum and minimum) and higher amplitude compared with NSD vessels. The frequency of lymphatic oscillation was better maintained in HSD rats than in NSD. Lymphatic pump efficiency including stroke volume index (SVI), frequency times SVI, and amplitude times frequency in HSD rats were significantly higher than those of NSD. Thus, a HSD enhances the resistance to pressure-induced decreases in lymphatic pump efficiency. The present ex vivo study suggest that collecting lymphatics of rats enhance myogenic activity and lymphatic pump efficiency to compensate for increase in lymph flow and/or pressure after 2 weeks salt loading.

High-Salt Diet Inhibits Expression of Angiotensin Type 2 Receptor in Resistance Arteries

Recent studies suggested that type 2 angiotensin receptor (AT2R) could contribute to regulation of blood pressure and/or vascular remodeling. A key question relates to the effects of potential modulators of vascular AT2R expression. In the present work, we evaluated if high salt intake (70 mmol/L NaCl in drinking water) could modulate rat mesenteric artery AT2R function and expression. Angiotensin II dose-response curves were studied in rat perfused pressurized small-diameter arteries in the presence of losartan (AT1R antagonist). Arteries were precontracted with phenylephrine, yielding approximately 30% decrease in resting diameter. AT2R activation by angiotensin-induced dose-dependent relaxation of precontracted arteries (60.1+/-9.1% of phenylephrine-induced contraction, P<0.05). In contrast, AT2R-dependent relaxation was not observed in arteries obtained from rats on high-salt diet. Semi-quantitative reverse-transcription polymerase chain reaction experiments demonstrated reduced amount of AT2R mRNA in arteries of rats on high-salt diet (65.5+/-7.5% of control levels, P<0.05). Western blot studies demonstrated decreased AT2R in mesenteric artery protein fractions of high-salt diet rats (60.0+/-18.0 of control levels, P<0.05). In a second set of experiments, adrenalectomy (4 days) blunted AT2R-mediated vasorelaxation and decreased AT2R mRNA (72.0+/-11.0% of control levels, P<0.05). AT2R abundance in protein fractions of mesenteric arteries of ADX rats was also diminished (64.0+/-13% of control levels, P<0.05). Both, AT2R mRNA and protein downregulation were prevented by mineralocorticoid replacement therapy. Finally, physiological concentrations of aldosterone caused a dose-dependent increase in AT2R mRNA of small diameter mesenteric artery explants. The results are consistent with aldosterone-mediated upregulation AT2R.

Nephron specific regulation of chloride channel CLC-K2 mRNA in the rat

This study investigated the influence of salt intake on the nephron specific gene expression of the kidney chloride channel CLC-K2. To this end, male Sprague-Dawley rats were fed a low (0.02% wt/wt), normal (0.6% wt/wt), or high salt (8% wt/wt) diet for ten days, or they received the loop diuretic furosemide (12 mg/kg/day) for six days. Expression and regulation of messenger RNA for CLC-K2 was demonstrated by RNase protection assay and in situ hybridization in kidney cortex, outer medulla and inner medulla. Tubular localization and regulation were determined precisely by reverse transcription-polymerase chain reaction (RT-PCR) and real time PCR of microdissected nephron segments. In situ hybridization analysis and RNase protection assay of the total kidney revealed a down-regulation of CLC-K2 mRNA in the high salt diet rats and an up-regulation of CLC-K2 mRNA in furosemide treated rats, which were restricted to the outer medulla. Microdissection of collagenase treated kidney revealed CLC-K2 mRNA expression in the outer medullary thick ascending limb (mTAL), cortical thick ascending limb (cTAL), distal convoluted tubule (DCT), connecting tubule and cortical collecting duct (CNT/CCD), and outer medullary collecting duct (OMCD), whereas no signals were detected in proximal convoluted and straight tubules (PCT and PST), descending thin limb from the outer medulla (dTL), descending and ascending thin limb from the inner medulla (TL), inner medullary collecting duct (IMCD) and glomeruli (glom). Using RT-PCR and real time PCR, the changing levels of CLC-K2 mRNA after furosemide treatment or high salt diet were restricted to the mTAL, whereas CLC-K2 mRNA levels in cTAL and OMCD were not changed in furosemide or high salt rats compared to time paired controls. Given that CLC-K2 expressed in the thick ascending limb of Henle's loop is responsible for net chloride reabsorption in this part of the nephron, our findings suggest that in states of surplus salt and in states of severe salt deprivation, selective regulation of CLC-K2 mRNA plays a role in the adaptation of the kidney to different salt loads.

Effects of valsartan on mechanical properties of the carotid artery in spontaneously hypertensive rats under high-salt diet.

The aim of this investigation was to evaluate the influence of a high-salt diet (HSD) on the effects of valsartan, an angiotensin II type 1 (AT(1)) receptor antagonist, on carotid arterial stiffness and structure in spontaneous hypertensive rats (SHR). Carotid arterial stiffness was studied in SHR receiving a HSD or a normal-salt diet (NSD) from the 10th to 20th week of age. Within each of the 2 groups, the animals received treatment with either placebo or valsartan (30 mg. kg(-1). d(-1)) administered on the 4th to 20th week of age. Arterial pressure, wall stress, incremental elastic modulus (Einc), medial cross-sectional area, and EIIIA fibronectin isoform were significantly increased in placebo-HSD rats compared with placebo-NSD rats with no change in the ratio of collagen to elastin. Valsartan reduced mean arterial pressure in both NSD and HSD rats but reduced pulse pressure only in NSD rats. In NSD rats, valsartan reduced Einc and medial cross-sectional area. In HSD, valsartan increased Einc and did not modify medial cross-sectional area and fibronectin. In valsartan-treated rats, the ratio of collagen to elastin was greater in HSD than in NSD rats. In conclusion, the effects of AT(1) blockade are greatly influenced by salt intake in SHR. Despite a reduction in mean arterial pressure in HSD rats, AT(1) blockade was not able to prevent the effects of a HSD on pulse pressure, carotid artery stiffness, and hypertrophy.

Isoform-specific regulation of nitric oxide synthase mRNA in the kidney by sodium and blood pressure.

The roles of nitric oxide synthases (NOS) in kidney function are still controversial, principally due to the lack of isoform-specific inhibitors of NOS. To investigate the relative roles of each isoform of NOS in regulation of sodium and volume homeostasis. We studied the effects of long-term modifications of sodium diet and blood pressure on expression of NOS mRNA in the renal cortex, where the three isoforms of NOS are present. We used quantitative reverse-transcription-polymerase chain reaction assays specific to each isoform of NOS to determine amounts of their respective mRNA in control rats, deoxycorticosterone acetate (DOCA)-salt hypertensive rats, rats fed a high-salt diet, and furosemide-treated rats fed a low-sodium diet. Nicotinamide adenine nucleotide phosphate H (NADPH) diaphorase staining was performed on DOCA-salt and control rat kidneys. Levels of NOS I mRNA in DOCA-salt rats were decreased by treatment, those in low-salt-diet rats remained unaffected and those in high-salt diet rats tended to be intermediate between those of the other rat groups. Expression of NOS III mRNA was not significantly modified by either treatment Levels of NOS II mRNA in DOCA-salt rats were increased, those in high-salt-diet rats remained unaffected, and those in low-salt-diet were decreased by treatment, but these levels are more than 100-fold lower than those observed for the other isoforms of NOS. NADPH diaphorase staining in macula densa of DOCA-salt rats was markedly decreased compared with that in macula densa of control rats but staining in renal inflammatory and fibrous lesions became detectable, and staining in the vessels did not differ from that for control rats. Our results show that intake of sodium and extracellular fluid volume regulate levels of mRNA of the three NOS isoforms in the renal cortex differently, suggesting that each of them plays a specific role.

Elevated vascular angiotensin converting enzyme mediates increased neointima formation after balloon injury in spontaneously hypertensive rats.

To measure the effect of hypertension on neointima formation after balloon injury of rat aorta and its association with the local angiotensin converting enzyme (ACE) concentration. Balloon angioplasty of the thoracic aorta using a 2 French Fogarty catheter was performed in spontaneously hypertensive rats (SHR) and normotensive Sprague-Dawley (SD) rats. The injured aortic wall of SHR had already significantly higher ACE concentrations than did the uninjured aortic wall of normotensive SD rats (media: 729 +/- 37 dpm/mm2 in SHR versus 496 +/- 38 dpm/mm2 in SD rats, P < 0.01; intima: 83 +/- 5 dpm/mm2 versus 68 +/- 6 dpm/mm2 in SD rats, P < 0.01). Fourteen days after injury of the aorta the hypertensive rats had significantly higher neointima: media ratios than did the normotensive rats (0.83 +/- 0.09 versus 068 +/- 0.01, P < 0.01). This was associated with a significant increase in vascular media and neointima ACE concentrations in SHR (media 965 +/- 25 dpm/mm2, neointima 614 +/- 48 dpm/mm2) compared with those in normotensive SD rats after balloon angioplasty (media 669 +/- 23 dpm/mm2, neointima 287 +/- 33 dpm/mm2, P < 0.01). ACE inhibitor treatment with 10 mg/kg body weight lisinopril daily for 14 days by gavage reduced neointima proliferation in hypertensive and normotensive rats (neointima: media ratio: 0.35 +/- 0.02 for SHR, P < 0.01, versus untreated SHR with balloon injury; 0.28 +/- 0.01 for SD, P < 0.01, versus untreated SD rats with balloon injury). This was associated with significant vascular media ACE inhibition (SHR 149 +/- 9 dpm/mm2; SD rats 118 +/- 7 dpm/mm2; P < 0.01 versus untreated controls with balloon injury) and neointima ACE inhibition (SHR 73 +/- 4 dpm/mm2, SD rats 63 +/- 7 dpm/mm2, P < 0.01, versus untreated controls with balloon injury), but also lowered the blood pressure in SHR significantly (to 148 +/- 5 mmHg, P < 0.01, versus untreated SHR with balloon injury). When this drop in blood pressure was prevented by feeding the rats a high-salt diet (SHR with ACE inhibitor plus high salt-diet group blood pressure 193 +/- 3 mmHg, P = 0.57, versus untreated SHR) hypertension per se without the local ACE increase (ACE concentration in SHR with ACE inhibitor high-salt diet rats' media 167 +/- 10 dpm/mm2 and neointima 81 +/- 9 dpm/mm2) had only a mild effect on neointima formation after balloon angioplasty (neointima: media ratio 0.4 +/- 0.01 for SHR with ACE inhibitor plus high-salt diet versus 0.35 +/- 0.02 for SHR with ACE inhibitor plus normal-salt diet P < 0.05). Treatment with 10 mg/kg body weight angiotensin II subtype 1 receptor antagonist losartan potassium daily for 14 days by gavage was associated with a reduction in neointima formation similar to that observed with the ACE inhibitor both for SHR and for SD rats (neointima: media ratio 0.32 +/- 0.04 for SHR with losartan, 0.27 +/- 0.03 for SD rats with losartan; P < 0.01, versus untreated controls with balloon injury) suggesting that ACE inhibitor prevented neointima formation, at least in part by, reducing the local production of angiotensin II. Neointima formation after balloon angioplasty in SHR is increased compared with that in normotensive SD rats. This is due mainly to there being a higher degree of activation of the renin-angiotensin system in the aorta of the SHR before and after balloon injury compared with that in normotensive SD rats measured in terms of the increased vascular ACE concentrations. Blood pressure alone had only a moderate effect on neointima formation.

Chronic manipulation of dietary salt modulates renal physiology and kidney dopamine receptor subtypes: Functional and autoradiographic studies

1. 1. Compared to rats maintained on the normal NaCI (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited increased plasma aldosterone and chloride and decreased urinary sodium excretion. 2. 2. Rats maintained on the high NaCl (8%) diet for 4 weeks showed increased systolic blood pressure, water intake, urine volume, sodium and dopamine excretion and decreased plasma aldosterone and glomerular filtration rate. 3. 3. Administration of SCH 23390 (10mg/kg, po), but not domperidone to the high salt diet rats attenuated the diuretic effect, indicating the involvement of DA 1 rather than DA 2 receptors. The dopamine decarboxylase inhibitor, carbidopa (30 mg/kg, i.p.), also reduced the high salt-induced diuresis. 4. 4. Kidney sections from rats fed the low NaCl diet showed a 63–100% decrease ( P < 0.001−0.02) in cortical and medullary DA 1 and DA 2 binding sites, while rats fed the high NaCl diet demonstrated only a 70% decrease ( P < 0.01−0.02) in cortical DA 1 binding, without affecting DA 2 binding. 5. 5. These data indicate that chronic modification of dietary salt profoundly affects the sodium, water and dopamine excretion and leads to selective modulation of renal dopamine receptor subtypes.