HS Diet Research Articles

Heterozygous knockout of transforming growth factor-β1 protects Dahl S rats against high salt-induced renal injury

The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-β1 (Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-β1(+/-) Dahl S). Intercrossing TGF-β1(+/-) rats did not produce any homozygous KO rats (66.4% +/-, 33.6% +/+), indicating that the mutation is embryonic lethal. Six-week-old wild-type (WT) littermates and TGF-β1(+/-) Dahl S rats were fed a 0.4% (low salt, LS) or 8% NaCl (high salt, HS) diet for 5 wk. Renal cortical expression of TGF-β1, urinary TGF-β1 excretion, proteinuria, glomerular injury and tubulointerstitial fibrosis, and systolic blood pressure were similar in WT and TGF-β1(+/-) Dahl S rats maintained on the LS diet. The expression and urinary excretion of TGF-β1 increased to a greater extent in WT than in TGF-β1(+/-)Dahl S rats fed an HS diet for 1 wk. Systolic blood pressure rose by the same extent to 235 ± 2 mmHg in WT and 239 ± 4 mmHg in TGF-β1(+/-) Dahl S rats fed a HS diet for 5 wk. However, urinary protein excretion was significantly lower in TGF-β1(+/-) Dahl S than in the WT animals. The degree of glomerular injury and renal cortical and outer medullary fibrosis was markedly less in TGF-β1(+/-) than in WT rats. These findings suggest that the loss of one copy of the TGF-β1 gene blunts the increase in renal TGF-β1 protein expression and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis in Dahl S rats fed an HS diet independently of changes in blood pressure.

Reduced ENaC activity and blood pressure in mice with genetic knockout of the insulin receptor in the renal collecting duct

To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male knockout (KO) mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to wild-type (WT) littermates. One week of LS feeding led to a significant reduction in urine potassium (K(+)) and sodium (Na(+)) excretion in KO, and a reduction in the ratio of Na(+) to chloride (Cl(-)) in plasma, relative to WT. HS diet (1 wk) increased plasma K(+) and reduced urine Na(+) to Cl(-) ratio in the KO. Furthermore, KO mice had a significantly (P = 0.025) blunted natriuretic response to benzamil, an epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (∼15%), lower band density for the β-subunit of ENaC in the KO vs. WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO vs. WT mice under basal conditions (mmHg): 112 ± 5 (WT), 104 ± 2 (KO), P = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homeostasis and BP.

Introgression of Brown Norway CYP4A genes on to the Dahl salt-sensitive background restores vascular function in SS-5BN consomic rats

The present study tested the hypothesis that the Dahl SS (salt-sensitive) rat has vascular dysfunction due, in part, to the up-regulation of the CYP4A/20-HETE (cytochrome P450 ω-hydroxylase 4A)/20-hydroxyeicosatetraenoic acid) system. To assess the role of vascular 20-HETE, SS rats were compared with SS-5(BN) consomic rats, carrying CYP4A alleles on chromosome 5 from the normotensive BN (Brown Norway) introgressed on to the SS genetic background. Cerebral arteries from SS-5(BN) rats had less CYP4A protein than arteries from SS rats fed either NS (normal-salt, 0.4% NaCl) or HS (high-salt, 4.0% NaCl) diet. ACh (acetylcholine)-induced dilation of MCAs (middle cerebral arteries) from SS and SS-5(BN) rats was present in SS-5(BN) rats fed on either an NS or HS diet, but absent in SS rats. In SS rats fed on either diet, ACh-induced dilation was restored by acute treatment with the CYP4A inhibitor DDMS (N-methyl-sulfonyl-12,12-dibromododec-11-enamide) or the 20-HETE antagonist 20-HEDE [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid]. The restored response to ACh in DDMS-treated SS rats was inhibited by L-NAME (N(G)nitro-L-arginine methyl ester) and unaffected by indomethacin or MS-PPOH [N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide]. Vascular relaxation responses to the NO donor C(5)FeN(6)Na(2)O were intact in both SS and SS-5(BN) rats and unaffected by the acute addition of DDMS, indicating that the vascular dysfunction of the SS rat is due to a reduced bioavailability of NO instead of failure of the VSMCs (vascular smooth muscle cells) to respond to the vasodilator. Superoxide levels in cerebral arteries of SS-5(BN) rats [evaluated semi-quantitatively by DHE (dihydroethidium) fluorescence] were lower than those in the arteries of SS rats. These findings indicate that SS rats have an up-regulation of the CYP4A/20-HETE pathway resulting in elevated ROS (reactive oxygen species) and reduced NO bioavailability causing vascular dysfunction.

Dietary sodium loading impairs microvascular function independent of blood pressure in humans: role of oxidative stress

Animal studies have reported dietary salt-induced reductions in vascular function independent of increases in blood pressure (BP). The purpose of this study was to determine if short-term dietary sodium loading impairs cutaneous microvascular function in normotensive adults with salt resistance. Following a control run-in diet, 12 normotensive adults (31 ± 2 years) were randomized to a 7 day low-sodium (LS; 20 mmol day(-1)) and 7 day high-sodium (HS; 350 mmol day(-1)) diet (controlled feeding study). Salt resistance, defined as a 5 mmHg change in 24 h mean BP determined while on the LS and HS diets, was confirmed in all subjects undergoing study (LS: 84 ± 1 mmHg vs. HS: 85 ± 2 mmHg; P > 0.05). On the last day of each diet, subjects were instrumented with two microdialysis fibres for the local delivery of Ringer solution and 20 mm ascorbic acid (AA). Laser Doppler flowmetry was used to measure red blood cell flux during local heating-induced vasodilatation (42°C). After the established plateau, 10 mm l-NAME was perfused to quantify NO-dependent vasodilatation. All data were expressed as a percentage of maximal cutaneous vascular conductance (CVC) at each site (28 mm sodium nitroprusside; 43°C). Sodium excretion increased during the HS diet (P < 0.05). The plateau % CVCmax was reduced during HS (LS: 93 ± 1 % CVCmax vs. HS: 80 ± 2 % CVCmax; P < 0.05). During the HS diet, AA improved the plateau % CVCmax (Ringer: 80 ± 2 % CVCmax vs. AA: 89 ± 3 % CVCmax; P < 0.05) and restored the NO contribution (Ringer: 44 ± 3 % CVCmax vs. AA: 59 ± 6 % CVCmax; P < 0.05). These data demonstrate that dietary sodium loading impairs cutaneous microvascular function independent of BP in normotensive adults and suggest a role for oxidative stress.

THE EFFECT OF SALT LOADING AND POTASSIUM SUPPLEMENT ON HYPERTENSION AND THE BALANCE OF TEFF AND TREG IN DAHL SALT SENSITIVE RATS

ObjectivesHigh dietary salt intake caused high blood pressure and renal injury Potassium had an antihypertensive effect. In recent years, the infiltration or activation of immune cells has been implicated in...

Abstract 6: Heterozygous TGFβ1 Zinc-Finger Knockout Dahl S Rats Reveal Protection Against High-Salt Induced Renal Injury

We used a zinc-finger nuclease strategy to create heterozygous TGFβ1 knockout rats (TGFβ1 +/- ) on a Dahl SS/Jr genetic background with a 22 base-pair frame shift mutation between nucleotides 191-212 which introduced a premature stop codon at amino acid 34. Intercrossing TGFβ1 +/- rats did not produce homozygous knockout rats, indicating that the mutation is embryonic lethal. Wildtype (WT) littermates and TGFβ1 +/- rats were fed either a 0.4% (normal salt, NS) or 8% NaCl (high salt, HS) diet for 5 weeks. When fed a NS diet, WT and TGFβ1 +/- exhibit similar renal cortical TGFβ1 expression (1.00±0.12 vs 1.05±0.05, arbitrary units), urinary TGFβ1 excretion (3.9±1.2 vs 5.3±0.4, μg/day), proteinuria (43±5 vs 36±4, mg/day), and minimal glomerular injury and tubulointerstitial fibrosis (TIF). 5 weeks of HS increased renal cortical TGFβ1 protein expression to a greater extent in WT versus TGFβ1 +/- (1.89±0.14 vs 1.52±0.09, arbitrary units) and TGFβ1 levels in urine increased to a greater extent in WT (41±10 μg/day) versus TGFβ1 +/- rats (18±4 μg/day) fed a HS diet for 1 week. Systolic blood pressure (SBP), measured by tail-cuff, was similar in WT (161±6 mmHg) and TGFβ1 +/- (162±7 mmHg) fed a NS diet and increased to the same extent in both WT (235±2 mmHg) and TGFβ1 +/- (239±4 mmHg) fed a HS diet for 5 weeks. Urinary protein excretion increased to a greater extent in WT versus TGFβ1 +/- (463±28 vs 313±36 mg/day) fed a HS diet for 5 weeks. Glomerular injury and renal cortical interstititial fibrosis were markedly reduced in TGFβ1 +/- versus WT after 5 weeks on a HS diet. Similarly, TIF in the renal medulla was less in TGFβ1 +/- compared with WT. These findings suggest that loss of one copy of the TGFβ1 gene blunts the increase in renal TGFβ1 protein in Dahl S rats fed a HS diet and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis independent of changes in blood pressure.

Bone marrow transplantation improves endothelial function in hypertensive Dahl salt-sensitive rats

Bone marrow-derived endothelial progenitor cells (EPCs) constitute an important endogenous system in the maintenance of endothelial integrity and vascular homeostasis. Cardiovascular risk factors are associated with a reduced number and functional capacity of EPCs. Here we investigated the effect of transplantation of bone marrow–derived cells from Dahl salt-resistant rat into age-matched Dahl salt-sensitive (DS) rat on blood pressure, endothelial function, and circulating EPC number. The recipient DS rats were fed a normal (0.5% NaCl, NS) or high-salt (4% NaCl, HS) diet for 6 weeks after bone marrow transplantation (BMT). DS rats on a NS or a HS diet without BMT were used as controls. Hypertensive DS (HS-DS) rat (systolic blood pressure: 213 ± 4 mm Hg vs. 152 ± 4 mm Hg in NS, P < .05) manifested impaired endothelium-dependent relaxation to acetylcholine (EDR), increased gene expression of vascular oxidative stress and proinflamamtory cytokines, and decreased eNOS expression. BMT on HS-DS rat significantly improved EDR and eNOS expression, reduced oxidative stress without reduction in SBP (206 ± 6 mm Hg). Flow cytometry analysis showed that there was no difference in the number of circulating EPCs, demonstrated by expression of EPC markers CD34, cKit, and vascular endothelial growth factor, between hypertensive and normotensive rats. Surprisingly, BMT resulted in a 5- to 10-fold increase in the previously mentioned EPC markers in hypertensive, but not normotensive rat. These results suggest that DS rat has an impaired ability to increase bone marrow–derived EPCs in response to HS diet challenge, which may contribute to endothelial dysfunction.

Abstract 446: Deletion of Il-6 Prevents Development of Cardiac Damage and Dysfunction in Chronic Ang Ii-salt-induced Hypertension

IL-6 knockout (KO) mice were reported to spontaneously develop cardiac dysfunction and fibrosis. These KO mice also develop less hypertension when fed high salt and infused with angiotensin II (Ang II). We tested the hypothesis that in IL-6-KO mice the attenuated hypertension in response to Ang II-salt is due to the development of cardiac dysfunction. Male C57Bl/6J and IL-6-/- mice (B6.129S6- Il6 tm1Kopf ) were implanted with telemetry devices for blood pressure measurements, infused with vehicle (V) or Ang II (90 ng/min/mouse subcutaneously) and feed a high salt diet (4% salt diet, HS) for 8 weeks (W). We studied 4 experimental groups: 1) C57BL/6J + V (n=9); 2) IL6-KO + V (n=9); 3) C57BL/6J + Ang II (n=8) and 4) IL6-KO + Ang II (n=6). Blood pressure and echocardiography data were collected before starting the HS diet and Ang II infusion (baseline) and 8 weeks after HS alone or combined with Ang II. Results (Mean±SEM) Conclusion: Our results do not support our hypothesis and shows that the lack of IL-6 does not affect development of hypertension or cardiac hypertrophy but rather prevents cardiac dysfunction, LV dilation, myocardial inflammation and fibrosis in Ang II-salt-induced hypertension, suggesting that IL-6 plays an important role in cardiac dysfunction associated with hypertension.

Blunted renal dopaminergic system in a mouse model of diet-induced obesity

Obesity has reached epidemic proportions in the Western world and is implicated in the pathophysiology of essential hypertension. The aim of the present study was to evaluate sodium handling, blood pressure and renal dopaminergic system activity in a mouse model of obesity induced by exposure to a hypercaloric diet. From six to 18 weeks of age, animals were fed with a control diet or a high-fat high-simple-carbohydrate (HFHSC) diet. Renal function, blood pressure and urinary and plasmatic catecholamines and biochemical parameters were evaluated in both groups. In parallel, the effects of high sodium intake (HS, 1.0% NaCl, 3 days) on natriuresis, urinary catecholamine excretion and aromatic l-amino acid decarboxylase (AADC) activity were evaluated in control and obese mice. Mice exposed to the HFHSC diet presented obesity, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia and increased blood pressure. This was accompanied, in obese mice, by decreases in urinary excretion of dopamine and metabolites as well as reduced AADC activity in renal tissues. During HS intake, absolute urinary dopamine excretion increased in control, but not in obese mice. This was accompanied in obese mice by a natriuretic resistance on day 1 of the HS diet. In addition, obese mice presented increased urinary and plasmatic noradrenaline levels, as well as an increased heart rate when compared with control mice. In conclusion, in this model of diet-induced obesity hyperinsulinemia, insulin resistance and increased sympathetic tone are associated with blunted renal dopaminergic activity. It is suggested that this may contribute to compromised sodium excretion and increased blood pressure in obesity.

Salt sensitivity of nitric oxide generation and blood pressure in mice with targeted knockout of the insulin receptor from the renal tubule

To elucidate the role of the insulin receptor (IR) on kidney nitric oxide generation and blood pressure (BP) control, we generated mice with targeted deletion of renal tubule IR using loxP recombination driven by a Ksp-cadherin promoter. Male knockout (KO) and wild-type (WT) littermates (~4 mo old) were transitioned through three 1-wk treatments: 1) low-NaCl diet (0.085%); 2) high-NaCl diet (HS; 5%); and 3) HS diet plus 3 mM tempol, a superoxide dismutase mimetic, in the drinking water. Mice were then switched to medium-NaCl (0.5%) diet for 5 days and kidneys harvested under pentobarbital anesthesia. Twenty-four-hour urinary nitrates plus nitrites were significantly higher in the WT mice under HS (2,067 ± 280 vs. 1,550 ± 230 nmol/day in WT and KO, respectively, P < 0.05). Tempol attenuated genotype differences in urinary nitrates plus nitrites. A rise in BP with HS was observed only in KO mice and not affected by tempol (mean arterial pressure, dark period, HS, 106 ± 5 vs. 119 ± 4 mmHg, for WT and KO, respectively, P < 0.05). Renal outer medullary protein levels of nitric oxide synthase (NOS) isoforms by Western blot (NOS1-3 and phosphorylated-S1177-NOS3) revealed significantly lower band density for NOS1 (130-kDa isoform) in the KO mice. A second study, when mice were euthanized under HS conditions, confirmed significantly lower NOS1 (130 kDa) in the KO, with an even more substantial (>50%) reduction of the 160-kDa NOS1 isoform. These studies suggest that the loss of renal IR signaling impairs renal nitric oxide production. This may be important in BP control, especially in insulin-resistant states, such as the metabolic syndrome.

Bitter Melon Seed Oil-Attenuated Body Fat Accumulation in Diet-Induced Obese Mice Is Associated with cAMP-Dependent Protein Kinase Activation and Cell Death in White Adipose Tissue1–3

The aim of this study was to investigate the antiadiposity effect of bitter melon seed oil (BMSO), which is rich in the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid. In Expt. 1, C57BL/6J mice were fed a butter-based, high-fat diet [HB; 29% butter + 1% soybean oil (SBO)] for 10 wk to induce obesity. They then continued to receive that diet or were switched to an SBO-based, high-fat diet alone (HS; 30% SBO) or containing bitter melon seed oil (BMSO) (HBM; 15% SBO + 15% BMSO) for 5 wk. The body fat percentage was significantly lower in mice fed the HBM diet (21%), but not the HS diet, compared with mice fed the HB diet. In Expt. 2, mice were fed an SBO-based, high-fat diet containing 0 (HS), 5 (LBM), 10 (MBM), or 15% (HBM) BMSO for 10 wk. In the LBM, MBM, and HBM groups, the body fat percentage was significantly lower by 32, 35, and 65%, respectively, compared with the HS control. The reduction in the HBM group was significantly greater than that in the LBM or MBM group. BMSO administration increased phosphorylation of acetyl-CoA carboxylase, cAMP-activated protein kinase (PKA), and signal transducer and activator of transcription 3 in the white adipose tissue (WAT), suggesting that PKA and leptin signaling might be involved in the BMSO-mediated reduction in lipogenesis and increase in thermogenesis and lipolysis. However, compared with the HS control, the HBM group had a significantly higher TNFα concentration in the WAT accompanied by TUNEL-positive nuclei. We conclude that BMSO is effective in attenuating body fat accumulation through mechanisms associated with PKA activation and programmed cell death in the WAT, but safety concerns need to be carefully addressed.

Effects of increased dietary sulfur on beef steer mineral status, performance, and meat fatty acid composition

Ninety-six crossbred yearling steers (321 ± 29 kg BW) were used to determine the effects of feeding cattle a high S diet on pasture before receiving a high S diet in the feedlot. Steers were blocked by BW, allocated to 2.4-ha bromegrass (Bromus inermis L.) pastures (n = 4 plots per treatment), and supplemented at 1% BW with either low S dried distillers grains with solubles (DDGS; 0.34% total diet S; LS) or LS DDGS with additional S (0.47% total diet S; HS) from NaSO(4) for 36 d. On d 37, steers moved into the feedlot where one-half remained on the previous S treatment and the other half switched treatments, resulting in 4 treatments (LS-LS, LS-HS, HS-LS, HS-HS; LS: 0.2 to 0.3% total diet S, HS: 0.5 to 0.6% total diet S; n = 6 feedlot pens per treatment). During the pasture period, forage mass offered, grazing residual mass, and in vitro digestible DM of forage did not differ among treatments (P > 0.40), and ADG did not differ (LS: 1.6 kg · d(-1), HS: 1.7 kg · d(-1), P = 0.54). Plasma Mg measured on d 35 was decreased by ≈ 5% in response to increased dietary S during the pasture period (P = 0.05), though no effect on plasma Mg was observed during finishing (P > 0.15). Plasma Cu concentrations on d 155 were ≈ 15% less (P = 0.02) in HS vs. LS steers, and d 155 liver Cu concentrations were ≈ 51% less in HS vs. LS steers (P = 0.01). Increased dietary S during the feedlot period decreased ADG by ≈ 10% (P = 0.01) and tended to decrease HCW by ≈ 5% (P = 0.06) compared with LS steers. Steers receiving the HS diet had increased stearic acid (C18:0) and heptadecanoic acid (C17:0; P = 0.04 and 0.01, respectively) percentages in rib facings collected at slaughter. Exposing cattle to greater S diets (0.47% S) during a forage-based diet did not influence later performance on high S feedlot diets (0.5 to 0.6% S); however, cattle fed high dietary S on pasture had greater fat cover at slaughter (P = 0.01), suggesting S may have influenced lipid metabolism.

Role of CYP4A/20‐HETE Pathway in Vascular Oxidative Stress in the Dahl Salt‐Sensitive Rat

We tested the hypothesis that cytochrome P450‐4A (CYP4A) and 20‐hydroxyeicosatetraenoic acid (20‐HETE) contribute to vascular oxidant stress and endothelial dysfunction in Dahl salt‐sensitive (SS) rats. Endothelial dysfunction in SS rats was ameliorated by a CYP4A inhibitor (DDMS) or a 20‐HETE antagonist (20‐HEDE), and in SS‐5BN consomic rats carrying CYP4A genes from the Brown Norway rat on the SS genetic background. Superoxide (SOX) levels, assessed via DHE fluorescence, were significantly higher in cerebral arteries of SS rats vs. SS‐5BN rats fed either normal salt (NS, 0.4% NaCl) or high salt (HS, 4.0% NaCl) diet. DDMS reduced SOX in arteries of HS‐fed SS rats to the same degree as a superoxide scavenger (PEG‐SOD), suggesting that the CYP4A/20‐HETE system contributes to elevated vascular SOX in HS‐fed SS rats. CYP4A protein expression was significantly higher in cerebral arteries of SS rats fed NS or HS diet vs. SS‐5BN rats. CuZn SOD, MnSOD, and EC‐SOD expression was similar in SS vs. SS‐5BN, indicating that the elevated vascular SOX in SS vs. SS‐5BN rats is not due to differences in antioxidant defenses. Treatment with the superoxide scavenger tempol had no effect on CYP4A expression in either strain. These findings indicate that increased expression/activity of the CYP4A/20‐HETE system contributes to vascular oxidant stress and endothelial dysfunction in SS rats fed either NS or HS diet. [NIH #HL65289, HL72920, HL92026, and AHA].

Effects of a high maternal sodium intake on offspring of genetically diverse (CCHS) mice

The present study sought to determine whether a high maternal salt intake during pregnancy and lactation affected offspring blood pressure (BP), heart rate (HR), or locomotor activity in a genetically diverse stock of mice (Collaborative Cross Heterozygous Stock, CCHS). Dams were fed a low or high salt diet (0.25% NaCl (LS) vs 5% NaCl (HS)) from before conception through weaning, and telemetry studies were performed in 10 month old male offspring raised on either the LS or HS diet. A maternal HS diet was associated with significantly smaller litter sizes (P=0.03, data from groups of 34 and 37 litters), but offspring weight was not affected at any time between the day of birth (data from 22 and 24 litters) and 10 months of age (group sizes ≥ 11). Telemetry studies revealed no effects of maternal HS on mean BP or HR. However, maternal HS was associated with increased circadian variation in BP, increased percentage of time spent active during the 12h light period, and a tendency towards increased pulse pressure (P=0.03, 0.04, 0.06, respectively, group sizes of 8 – 14). Our results indicate that a maternal high salt diet does not affect offspring mean BP level in 10 month old male CCHS mice, but does reduce litter size and has subtle effects on activity and blood pressure cycles. Supported by the Canadian Institutes of Health Research (#91547).

Salt‐Dependent Hypertension and Renal Injury are Associated with Increased Excretion of Angiotensinogen and Angiotensin‐ (1‐12) in Female mRen2.Lewis Rats

The female mRen2.Lewis (mRen2) congenic strain exhibits a pronounced sensitivity to salt in regards to blood pressure, oxidative stress, and renal injury. However, the expression of renin within the kidney is suppressed in the mRen2 which likely reflects the intact response to the higher blood pressure. The present study examined the renal response to a high sodium (HS, 4% Na) diet in female mRen2 (15 wks of age) regarding the urinary components of the renal RAS. In comparison to normal salt (0.5% Na, NS), the HS diet markedly increased systolic blood pressure [224 ± 8 vs. 149 ± 5 mmHg, P&lt;0.01] and proteinuria [98 ± 9 vs. 7 ± 1 mg/kg/day, P&lt;0.01]. Urinary excretion of angiotensinogen (Aogen) increased almost 200‐fold in the HS mRen2 [1.8 ± 0.2 vs. 0.01 ± 0.001 μg/kg/day, P&lt;0.01]. Urinary levels of Ang II and Ang‐(1‐7) increased 1.4‐fold and 3.6‐fold, respectively in the HS mRen2. In contrast, excretion of the novel peptide Ang‐(1‐12) increased over 40‐fold in the HS rats and more closely reflects the increase in the Aogen precursor. Extended HPLC separation revealed a single immunoreactive peak of Ang‐(1‐12) in pooled urine extracts from NS and HS mRen2 that eluted at a position distinct from Ang‐(1‐7), Ang II or Ang I. We conclude that active processing of Aogen to Ang‐(1‐12) may contribute to the salt‐sensitive phenotype of the female mRen2 and may reflect a renin‐independent pathway within the kidney. Funding: HL‐56973, HL‐51952.

Acute dietary salt loading impairs cutaneous microvascular function in normotensive salt‐resistant adults: role of oxidative stress

Animal studies have reported dietary salt‐induced reductions in vascular function without an increase in blood pressure (BP). The purpose of this study was to determine if acute dietary salt loading impairs cutaneous microvascular function in normotensive adults with salt‐resistant BP. Following a control run‐in diet, 10 normotensive salt‐resistant subjects (31±3 yr) were randomized to a 7 day high salt (HS; 350 mmol/day) and 7 day low salt (LS; 20 mmol/day) diet (controlled feeding study). Salt‐resistant BP was defined as a change in 24‐hour mean BP of ≤ 5 mmHg from the LS to the HS diet. On the last day of each diet, subjects were instrumented with three microdialysis fibers for the local delivery of Ringer's solution (R), 10 mM L‐NAME, and 20 mM ascorbic acid (AA). Laser Doppler flowmetry was used to measure red blood cell flux during local heating (42□). All data were expressed as a percentage of the maximum cutaneous vascular conductance (CVC) at each site (28 mM SNP; 43□). The plateau %CVCmax was reduced during HS (LS 93±1 v. HS 80±2, P&lt;0.05), as was the nitric oxide (NO) contribution to the plateau (LS 55±5 v. HS 46±3, P=0.075). During HS, AA improved the plateau %CVCmax (R 80±2 v. AA 89±3, P&lt;0.05) and restored the NO contribution (R 46±3 v. AA 60±7, P&lt;0.05). These data suggest that acute dietary salt loading impairs cutaneous microvascular function in normotensive salt‐resistant adults and indicate a role for oxidative stress.Supported by 2 P20 RR016472‐11 and HL104106.

The type and quantity of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome ‘at-risk’ population

An obese-type human microbiota with an increased Firmicutes:Bacteroidetes ratio has been described that may link the gut microbiome with obesity and metabolic syndrome (MetS) development. Dietary fat and carbohydrate are modifiable risk factors that may impact on MetS by altering the human microbiome composition. We determined the effect of the amount and type of dietary fat and carbohydrate on faecal bacteria and short chain fatty acid (SCFA) concentrations in people 'at risk' of MetS. A total of 88 subjects at increased MetS risk were fed a high saturated fat diet (HS) for 4 weeks (baseline), then randomised onto one of the five experimental diets for 24 weeks: HS; high monounsaturated fat (MUFA)/high glycemic index (GI) (HM/HGI); high MUFA/low GI (HM/LGI); high carbohydrate (CHO)/high GI (HC/HGI); and high CHO/low GI (HC/LGI). Dietary intakes, MetS biomarkers, faecal bacteriology and SCFA concentrations were monitored. High MUFA diets did not affect individual bacterial population numbers but reduced total bacteria and plasma total and LDL-cholesterol. The low fat, HC diets increased faecal Bifidobacterium (P=0.005, for HC/HGI; P=0.052, for HC/LGI) and reduced fasting glucose and cholesterol compared to baseline. HC/HGI also increased faecal Bacteroides (P=0.038), whereas HC/LGI and HS increased Faecalibacterium prausnitzii (P=0.022 for HC/HGI and P=0.018, for HS). Importantly, changes in faecal Bacteroides numbers correlated inversely with body weight (r=-0.64). A total bacteria reduction was observed for high fat diets HM/HGI and HM/LGI (P=0.023 and P=0.005, respectively) and HS increased faecal SCFA concentrations (P<0.01). This study provides new evidence from a large-scale dietary intervention study that HC diets, irrespective of GI, can modulate human faecal saccharolytic bacteria, including bacteroides and bifidobacteria. Conversely, high fat diets reduced bacterial numbers, and in the HS diet, increased excretion of SCFA, which may suggest a compensatory mechanism to eliminate excess dietary energy.

Effect of replacing barley with corn or sorghum grain on rumen fermentation characteristics and performance of Iranian Baluchi lamb fed high concentrate rations

The present study, conducted in Iran, was aimed to evaluate the effects of partial substitution of barley with corn or sorghum (slowly fermenting grains) on in situ dry matter (DM) and starch degradability and in vivo digestion characteristics and performance in finishing Iranian Baluchi lambs. In the first experiment, samples of ground barley (B), corn (C), sorghum (S) and their combinations [%: 70 barley : 30 corn (LC), 70 barley : 30 sorghum (LS), 30 barley : 70 corn (HC) and 30 barley : 70 sorghum (HS)] were incubated in nylon bags in rumen of three fistulated Holstein steers. Effective degradability (ED) for DM and starch were different (P &lt; 0.05) among treatments. In both cases, ED was higher (P &lt; 0.05) for B than that for C, S, HC and HS. Results of the present study confirmed previous reports that ruminal degradability of barley is higher than that of corn and sorghum, and further suggested that there may be benefits in replacing barley with ~70% of corn or sorghum. In Experiment 2, 30 Iranian Baluchi male lambs weaned at 65 days of age (BW 32 ± 2.3 kg) were divided into five equal weight groups (6 animals per group) in a complete randomised design. Lambs received a mixed diet in the form of total mixed rations (TMR), consisting of 32.5% alfalfa hay [89% DM, 43% natural detergent fibre (NDF) and 16% crude protein (CP) in diet DM basis] and 67.5% of the respective concentrates. The five iso-caloric (10.73 MJ ME/kg) and iso-nitrogenous (14% CP/kg DM) concentrates with different sources of starch used in the study were (1) barley, as the control cereal source for starch, and in the other treatments barley substituted with (2) 30% corn (LC), (3) 30% sorghum (LS), (4) 70% corn (HC) and (5) 70% sorghum (HS). Lambs in HC and HS diets recorded increases of 20 and 50 g/day in average daily gain, respectively, compared with lambs in B. Substitution of B with 70% C and S significantly (P &lt; 0.01) improved ruminal pH and, presumably, resulted in higher starch outflow for post-ruminal digestion, as reflected by a higher blood glucose concentration, particularly for lambs in the HS diet. Results of the present study reaffirmed the advantages of feeding a mixture of grains with differing fermentation rates and, as previously reported, also suggested that the inclusion rate of slowly degradable grains should be ~70% so as to enhance animal performance.

Histone demethylase LSD1 deficiency during high-salt diet is associated with enhanced vascular contraction, altered NO-cGMP relaxation pathway, and hypertension

Histone methylation, a determinant of chromatin structure and gene transcription, was thought to be irreversible, but recent evidence suggests that lysine-specific demethylase-1 (LSD1, Kdm1a) induces demethylation of histone H3 lysine 4 (H3K4) or H3K9 and thereby alters gene transcription. We previously demonstrated a human LSD1 phenotype associated with salt-sensitive hypertension. To test the hypothesis that LSD1 plays a role in the regulation of blood pressure (BP) via vascular mechanisms and gene transcription, we measured BP and examined vascular function and endothelial nitric oxide (NO) synthase (eNOS) expression in thoracic aorta of male wild-type (WT) and heterozygous LSD1 knockout mice (LSD1(+/-)) fed either a liberal salt (HS; 4% NaCl) or restricted salt diet (LS; 0.08% NaCl). BP was higher in LSD1(+/-) than WT mice on the HS diet but not different between LSD1(+/-) and WT mice on the LS diet. Further examination of the mechanisms of this salt-sensitive hypertension in LSD1(+/-) mice on the HS diet demonstrated that plasma renin activity and plasma levels and urinary excretion of aldosterone were less in LSD1(+/-) than WT, suggesting suppressed renin-angiotensin-aldosterone system. In contrast, phenylephrine (Phe)-induced aortic contraction was greater in LSD1(+/-) than WT mice on the HS diet. Treatment of aortic rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a blocker of guanylate cyclase) enhanced Phe contraction in LSD1(+/-) compared with WT mice on the HS diet. Acetylcholine (Ach)-induced relaxation was less in LSD1(+/-) than WT mice on the HS diet. Endothelium removal or pretreatment with N(ω)-nitro-L-arginine methyl ester (blocker of NOS) or ODQ abolished Ach-induced relaxation in aorta of WT but had minimal effect in LSD1(+/-). Vascular relaxation to sodium nitroprusside, an exogenous NO donor and guanylate cyclase activator, was decreased in LSD1(+/-) vs. WT mice on the HS diet. RT-PCR and Western blots revealed decreased eNOS mRNA expression and eNOS and guanylate cyclase protein in the heart and aorta of LSD1(+/-) compared with WT mice on HS diet. Thus, during the HS diet, LSD1 deficiency is associated with hypertension, enhanced vascular contraction, and reduced relaxation via NO-cGMP pathway. The data support a role for LSD1-mediated histone demethylation in the regulation of NOS/guanylate cyclase gene expression, vascular function, and BP during the HS diet.

Estrogen Receptor GPR30 Reduces Oxidative Stress and Proteinuria in the Salt-Sensitive Female mRen2.Lewis Rat

The current study assessed whether activation of the novel estrogen receptor GPR30 ameliorates salt-dependent renal damage in intact mRen2.Lewis (mRen2) females. Hemizygous mRen2 rats were maintained on either a normal salt (0.5% Na) or high-salt (HS; 4.0% Na) diet for 10 weeks (5 to 15 weeks of age), and HS animals were treated with the GPR30 agonist G-1 or vehicle for 2 weeks. Systolic blood pressure markedly increased with HS diet (149±3 to 219±5 mm Hg; P<0.01), but G-1 did not influence pressure (P=0.42). G-1 and estradiol induced relaxation of preconstricted mesenteric vessels from normal salt mRen2 rats, but both responses were attenuated in the HS group. Despite the lack of an effect on blood pressure, G-1 decreased renal hypertrophy, proteinuria, urinary 8-isoprostane excretion, and tubular 4-hydroxynonenal staining. HS diet significantly increased GPR30 mRNA (1.01±0.04 versus 1.59±0.13; P<0.01) and protein (0.60±0.31 versus 3.99±0.75; P<0.01) in the renal cortex. GPR30 was highly expressed in the brush border of proximal tubules and colocalized with megalin. Finally, megalin expression was reduced by HS diet and restored with G-1. We conclude that GPR30-mediated beneficial effects in salt-sensitive mRen2 females occurred independent of changes in systolic blood pressure. The failure of G-1 to influence pressure may reflect a salt-induced impairment in GPR30-mediated vasorelaxation. The renoprotective actions of GPR30 may involve attenuation of tubular oxidative stress and activation of megalin-mediated protein reabsorption.