Salt Diet Group Research Articles

The underlying mechanisms of DNA methylation in high salt memory in hypertensive vascular disease

This study demonstrates the effect and DNA methylation-related mechanisms of a high-salt diet and salt memory-induced hypertension and vasculopathy. Thirty Sprague Dawley rats were randomly divided into a control (CON) group (n = 6) and a modeling group (n = 24). A 12% NaCl solution (1 mL/100 g) was intragastrically administered for 60 consecutive days for modeling. An increase in blood pressure up to 140 mmHg was considered successful modeling. Twelve of fifteen successfully modeled rats were randomly selected and divided into a High Salt Diet (HSD) group and a High Salt Memory (HSM) group (n = 6). Rats in HSD group were intragastrically administered a 12% NaCl solution, while rats in HSM group were administered a 3% NaCl solution twice a day for 30 days. At the end of the intervention, blood pressure and the serum levels of ET-1, NO, TNF-α and IL-1β were measured. RRBS-heavy sulfite sequencing technology was selected for DNA methylation analysis. The systolic blood pressure of rats in the HSD group and HSM group was significantly higher than that in the CON group. Compared with those in the CON group, the serum levels of ET-1 in the HSM group and the serum levels of NO in the HSD group and HSM group were significantly increased. The methylation level of the CON group was lower than that of the HSD group and the HSM group, and there was no significant difference between the HSD group and the HSM group. The methylation level of Myoz3 was downregulated in the HSD group and HSM group. The methylation level of Fgd3 were upregulated in HSD group and downregulated in the HSM group. The methylation levels of AC095693.1, Adamts3, PDGFA and PDGFRα were downregulated in the HSD group and upregulated in the HSM group. According to the GO database, the differentially methylated genes were significantly enriched in the coordination of cell function, genetic development, and RNA transcription. There were three main metabolic pathways that were enriched in the differentially expressed genes between the groups: the PI3K-Akt signaling pathway, MAPK signaling pathway, and Hippo signaling pathway. Excessive salt intake may cause hypertension and vascular damage, and this damage may continue after the reduction of salt intake. Therefore, salt memory phenomenon exists, and this memory effect may be correlated with the levels of DNA methylation.

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.

Common salt aggravated pathology of testosterone-induced benign prostatic hyperplasia in adult male Wistar rat

BackgroundBenign prostatic hyperplasia (BPH) is a major health concern associated with lower urinary tract symptoms and sexual dysfunction in men. Recurrent inflammation, decreased apoptotic rate and oxidative stress are some of the theories that explain the pathophysiology of BPH. Common salt, a food additive, is known to cause systemic inflammation and redox imbalance, and may serve as a potential risk factor for BPH development or progression. This study examined the effect of common salt intake on the pathology of testosterone-induced BPH.MethodsForty male Wistar rats were randomly divided into four equal groups of 10: a control and three salt diet groups-low-salt diet (LSD), standard-salt diet (SSD) and high-salt diet (HSD). The rats were castrated, allowed to recuperate and placed on salt-free diet (control), 0.25% salt diet (LSD), 0.5% salt diet (SSD) and 1.25% salt diet (HSD) for 60 days ad libitum. On day 33, BPH was induced in all the rats with daily injections of testosterone propionate-Testost® (3 mg/kg body weight) for 28 days. The rats had overnight fast (12 h) on day 60 and were euthanized the following day in order to collect blood and prostate samples for biochemical, molecular and immunohistochemistry (IHC) analyses. Mean ± SD values were calculated for each group and compared for significant difference with ANOVA followed by post hoc test (Tukey HSD) at p < 0.05.ResultsThis study recorded a substantially higher level of IL-6, IL-8 and COX-2 in salt diet groups and moderate IHC staining of COX-2 in HSD group. The prostatic level of IL-17, IL-1β, PGE2, relative prostate weight and serum PSA levels were not statistically different. The concentrations of IGF-1, TGF-β were similar in all the groups but there were multiple fold increase in Bcl-2 expression in salt diet groups-LSD (13.2), SSD (9.5) and HSD (7.9) and multiple fold decrease in VEGF expression in LSD (-6.3), SSD (-5.1) and HSD (-14.1) compared to control. Activity of superoxide dismutase (SOD) and concentration of nitric oxide rose in LSD and SSD groups, and SSD and HSD groups respectively. Activities of glutathione peroxidase and catalase, and concentration of NADPH and hydrogen peroxide were not significantly different. IHC showed positive immunostaining for iNOS expression in all the groups while histopathology revealed moderate to severe prostatic hyperplasia in salt diet groups.ConclusionsThese findings suggest that low, standard and high salt diets aggravated the pathology of testosterone-induced BPH in Wistar rats by promoting inflammation, oxidative stress, while suppressing apoptosis and angiogenesis.

Evaluation of Lipid Profile of High Salt fed Rats treated with L-Arginine

There is a global concern on salt consumption above the dietary guideline; salt consumption evokes physiological responses with cardiovascular risks associated with dyslipidemia other than increased blood pressure as numerous studies have pointed out. This study aimed at evaluating the effect of L-Arginine on lipid profile of rats fed high salt diet. Forty Male Albino Wister rats weighing between 70-120g were randomly selected assigned into four groups of 10 rats each. Group 1 served as the control and was given distilled water and normal rat chow. Group 2 was fed with high salt diet (8% Nacl in feed, and 1% Nacl in drinking water) Group 3 was treated as group 2 with the introduction of L-Arginine on the 43rd day of the experiment. Group 4 was treated as group 2 with the introduction of losartan administration on the 43rd day of the experiment. Administration of L-Arginine and losartan lasted for 14 days, making a total duration of feeding and drugs administration 56 days. At the end of the 56th day, the rats were fasted overnight for 12 hours and sacrificed under anaesthesia using sodium pentobarbitone. Blood samples were then collected from each animal via cardiac puncture into heparinized tubes and centrifuged at 3500rpm for a period of 15 min, and the clear supernatant plasma were collected and stored at -20°C for biochemical analyses of lipid profile. The results showed a significant increase in TG, LDL-C, TC, VLDL-C and a reduction of HDL-C in the salt fed group. Conversely, a significant reduction in TG, LDL-C, TC, VLDL-C and an increase in HDL-C was shown in the salt + L-Arginine treated group when compared to the control. The changes observed in the L-Arginine treated groups reversed the hyperlipidemia in the salt treated group which indicates L-Arginine is beneficial in treatment of salt induced dyslipidemia and cardiovascular diseases. Keywords: cardiovascular risks, dyslipidemia, L-Arginine, rats fed high salt diet

High Salt Diet Impairs Male Fertility in Mice via Modulating the Skeletal Homeostasis.

Male reproductive functions and bone health are both adversely affected by the high salt diet (HSD). Nevertheless, the underlying mechanism via which it alters the sperm function remains largely unknown. This study examines the mechanism by which HSD affects male fertility by impairing bone health. For investigating the same, male BALB/c mice were categorized into three groups-HSD group (fed with 4% NaCl), a low salt diet (LSD) group (fed with 0.4% NaCl), and a control group (fed with a normal diet) for 6 weeks and thereafter assessed for various sperm parameters, bone turnover markers, and testosterone levels. Furthermore, the quantitative assessment of testosterone biosynthesis enzymes was performed. Interestingly, we observed that mice fed with HSD showed significant alterations in sperm parameters-motility, count, and vitality, including morphological changes compared to both the LSD and the control groups. In addition, serum analysis showed an increase in bone resorption markers and a decrease in bone formation markers in the HSD group (p < 0.05). Further, HSD caused a decrease in the testosterone level and mRNA expression of testosterone biosynthesis enzymes. Importantly, a significant decrease in bone formation marker osteocalcin (OC) was observed to coincide with the dip in testosterone level in the HSD group. Given that OC plays a key role in maintaining male fertility, the above findings suggest that a decrease in OC levels may affect the testosterone biosynthesis pathway, reducing testosterone hormone secretion and thereby resulting in decreased spermatogenesis. The study for the first time delineates and bridges the mechanism of HSD-mediated bone loss (results in a deficiency of OC) with decreased testosterone biosynthesis and thus impaired male fertility.

The Effects of Dietary Salt on Blood Pressure Variability, Cerebral Perfusion and Cognitive Function in Young-Adult Male Rats

Background: There is controversy about the relationship between dietary salt intake and cognitive function. Experimental studies showed that extremely high salt diets increased blood pressure (BP) levels, leading to cognitive impairment. However, extremely high salt diets are not clinically relevant. The present study examined how high dietary salt could affect cognitive function and whether these effects are via BP stability and cerebral hypoperfusion. Methods: SD rats (male, young adult) were on the control diet (0.4% NaCl), low salt diet (0.1% NaCl), or high salt diets (with three does at 1%, 2%, or 4% NaCl) for 12 weeks. BP levels, heart rate (HR), and BP variability (SD and CV) were measured repeatedly at baseline, 1, 2, 3, 4, 6, 8, and 12 weeks after the onset of the diet. Anxiety and cognitive function (open field, novel object recognition, and T-maze) were measured repeatedly at baseline, 4, 8, and 12 weeks after the onset of the diet. Blood Na+ and cerebral blood flow (CBF) were measured at the endpoint. Results: Rats with the low salt diet showed significantly reduced systolic BP (SBP) and diastolic BP (DBP) levels compared to those with the control diet and high salt diets at 8 weeks after the onset of the diet. Notably, rats with the low salt diet had the highest SBP variability (SBPV) and DBPV, while those with the control diet showed the lowest SBPV and DBPV. In high salt diet groups, SBPV and DBPV increased in a dose-dependent manner. There is no significant difference in HR between groups. Rats only with a 4% NaCl diet showed worse cognitive function compared to rats with low salt and control diet 8 weeks after the onset. Blood Na+ was similar between groups. Interestingly, the high salt diet increased the CBF in a dose-dependent manner. The CBF of the low salt diet was similar to those with 2% salt die, and the CBF of the control group was the lowest. Conclusions: This study suggests that a moderate-high salt diet would not affect cognitive function in young-adult male SD rats. Only consumption of an extremely high salt diet impaired cognitive function, and this harmful effect was not via cerebral hypoperfusion. Moreover, both low and high salt diets increased BPV. This research was funded by the START-UP FUNDING OF HANGZHOU NORMAL UNIVERSITY, grant number 4255C50221204123 (Dr. Fen Sun). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Salt-sensitive hypertension after reversal of unilateral ureteral obstruction

The incidence of ureter obstruction is increasing and patients recovering from this kidney injury often progress to chronic kidney injury. There is evidence that a long-term consequence of recovery from ureter obstruction is an increased risk for salt-sensitive hypertension. A reversal unilateral ureteral obstruction (RUUO) model was used to study long-term kidney injury and salt-sensitive hypertension. In this model, we removed the ureteral obstruction at day 10 in mice. Mice were divided into four groups: (1) normal salt diet, (2) high salt diet, (3) RUUO normal salt diet, and (4) RUUO high salt diet. At day 10, the mice were fed a normal or high salt diet for 4 weeks. Blood pressure was measured, and urine and kidney tissue collected. There was a progressive increase in blood pressure in the RUUO high salt diet group. RUUO high salt group had decreased sodium excretion and glomerular injury. Renal epithelial cell injury was evident in RUUO normal and high salt mice as assessed by neutrophil gelatinase-associated lipocalin (NGAL). Kidney inflammation in the RUUO high salt group involved an increase in F4/80 positive macrophages; however, CD3+ positive T cells were not changed. Importantly, RUUO normal and high salt mice had decreased vascular density. RUUO was also associated with renal fibrosis that was further elevated in RUUO mice fed a high salt diet. Overall, these findings demonstrate long-term renal tubular injury, inflammation, decreased vascular density, and renal fibrosis following reversal of unilateral ureter obstruction that could contribute to impaired sodium excretion and salt-sensitive hypertension.

High Dietary Salt Intake Is Associated With Histone Methylation in Salt-Sensitive Individuals

BackgroundHigh salt diet is one of the important risk factors of hypertension and cardiovascular diseases. Increasingly strong evidence supports epigenetic mechanisms' significant role in hypertension. We aimed to explore associations of epigenetics with high salt diet, salt sensitivity (SS), and SS hypertension.MethodsWe conducted a dietary intervention study of chronic salt loading in 339 subjects from northern China in 2004 and divided the subjects into different salt sensitivity phenotypes. A total of 152 participants were randomly selected from the same cohort for follow-up in 2018 to explore the effect of a high-salt diet on serum monomethylation of H3K4 (H3K4me1), histone methyltransferase Set7, and lysine-specific demethylase 1 (LSD-1).ResultsAmong SS individuals, the blood pressure (SBP: 140.8 vs. 132.9 mmHg; MAP: 104.2 vs. 98.7 mmHg) and prevalence of hypertension (58.8 vs. 32.8%) were significantly higher in high salt (HS) diet group than in normal salt (NS) diet group, but not in the salt-resistant (SR) individuals (P > 0.05). Serum H3K4me1 level (287.3 vs. 179.7 pg/ml, P < 0.05) significantly increased in HS group of SS individuals, but not in SR individuals. We found daily salt intake in SS individuals was positively correlated with serum H3K4me1 (r = 0.322, P = 0.005) and Set7 (r = 0.340, P = 0.005) levels after adjusting for age and gender, but not with LSD-1 (r = −0.137, P = 0.251). In addition, positive correlation between the serum H3K4me1 level and Set7 level (r = 0.326, P = 0.007) was also found in SS individuals. These correlations were not evident in SR individuals.ConclusionOur study indicates that high salt diet increases the serum H3K4me1 and Set7 levels in SS individuals.

Low-dose angiotensin II supplementation restores flow-induced dilation mechanisms in cerebral arteries of Sprague-Dawley rats on a high salt diet.

Salt-induced suppression of angiotensin II contributes to impaired endothelium-dependent vascular reactivity. The present study investigated the effect of chronic low-dose angiotensin II (ANG II) supplementation on the mechanisms of flow-induced dilation (FID) and oxidative stress at the cellular and molecular level in middle cerebral arteries (MCA) of male Sprague-Dawley rats fed high salt diet. Rats (10 weeks old) were randomly assigned to a low salt diet group (0.4% NaCl in rat chow); high salt diet group (7 days 4% NaCl in rat chow) or HS+ANG II group [7 days high salt diet with 3 days ANG II administration via osmotic minipumps (100 ng/kg per min on days 4-7)]. FID was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenase (COX-1,2) inhibitor indomethacin, a selective inhibitor of CYP450 epoxygenase activity (MS-PPOH) and the superoxide dismutase mimetic TEMPOL. Gene expression of antioxidative enzymes, and of genes and proteins involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular nitric oxide and superoxide/reactive oxygen species levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters were measured by spectrophotometry. Chronic low-dose ANG II supplementation in high salt fed rats restored FID of MCAs, which was nitric oxide, prostanoid and epoxyeicosatrienoic acid dependent. ANG II changed the protein/gene expression of COXs, HIF-1α and VEGF and significantly increased GPx4 and EC-SOD antioxidative enzyme expression, decreased systemic oxidative stress, decreased superoxide/ROS levels and increased nitric oxide bioavailability in the vascular wall. Physiological levels of circulating ANG II are crucial to maintain the HIF-1α dependent mechanisms of FID and vascular oxidative balance without affecting mean arterial pressure.

Renocardiac protective effects of SGLT2 inhibitor combined with angiotensin receptor blocker in salt sensitive Dahl rats

Abstract Background Kidney plays a central role in regulating salt-sensitivity of blood pressure (BP) to governs sodium excretion via several mechanisms including pressure natriuresis and the actions of renal sodium transporters. Purpose We clarified the effects of combination treatment of sodium-glucose cotransporter 2 (SGLT2) inhibitor and angiotensin receptor blocker (ARB) on BP and the pathogenesis of renocardiac injuries, and elucidated underlying molecular mechanisms involved in the regulation of renal sodium handling in the development of salt-sensitivity by comparing with each monotreatment in Dahl salt-sensitive (DSS) hypertensive rats. Methods DSS rats were treated orally for 8-weeks with normal salt diet (0.3% NaCl) (NS/Cont group), high salt diet (8% NaCl) (HS/Cont group), high salt diet with ipragliflozin (0.04%) (HS/Ipra group), high salt diet with losartan (0.05%) (HS/Los group), or high salt diet with combination of ipragliflozin and losartan (HS/Ipra+Los group). Results The combination group significantly reduced systolic BP compared with either high salt diet control group, losartan or ipragliflozin monotreatment groups (HS/Ipra+Los: 182.5±18.4mmHg vs HS/Cont: 227.7±26.1; HS/Ipra: 216.6±26.9; HS/Los: 208.6±21.6, at 8-weeks of treatment, P&amp;lt;0.05, respectively) (Figure 1A). The slope of pressure-natriuresis curve was significantly increased in the HS/Ipra+Los group compared to that in the HS/Cont group (interaction P=0.024), HS/Ipra group (P=0.009), and HS/Los group (P=0.084) using the linear regression model (Figure 1B), which indicated that only the combination treatment of ipragliflozin and losartan improved salt-sensitivity. The combined treatment significantly improved creatinine clearance (HS/Ipra+Los: 3.3±0.9mL/min vs HS/Cont: 1.1±0.5; HS/Ipra: 1.7±0.6; HS/Los: 1.9±0.8, P&amp;lt;0.05, respectively). The combination treatment also significantly ameliorated glomerulosclerosis, and improved cardiomyocyte hypertrophy and perivascular fibrosis (Figure 1C). Angiotensin II type 1 receptor (AT1R) protein expression level in the kidney was remarkably suppressed in the combination treatment group compared to the other high salt diet groups. The protein expression level of Na+/H+ exchanger isoform 3 (NHE3) and Na+-K+-Cl– cotransporter 2 (NKCC2), two of major sodium transports in the renal tubules, were significantly decreased with losartan monotreatment and combination treatment, but not with ipragliflozin monotreatment (Figure 2). Conclusions The dual inhibition of SGLT2 and AT1R effectively improved salt-sensitivity via reducing renal expression levels of the sodium transporters, which eventually lead to renocardiac protection. Thus, the combination treatment could be a novel and useful therapeutic strategy for treating salt-sensitive hypertension and renal injury in non-diabetic patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research

RENALASE PROTECTS AGAINST SALT-INDUCED KIDNEY INJURY AND INFLAMMATION BY INHIBITING THE ACTIVATION OF THE AKT-1 SIGNALING PATHWAYS

Objective: An excess of dietary salt is the most common environmental factor that contributes to the development of hypertension leading to renal pathologies. Renalase (RNLS), a novel secreted flavoprotein with oxidoreductase activity, functions as a survival factor and promotes cell and organ survival. Previous evidence showed that recombinant renalase exerts powerful hypotensive effects and ameliorates renal interstitial fibrosis in rodents. In this study, we evaluated the potential protective role of renalase and underlying mechanisms in salt-induced kidney injury and inflammation. Design and method: In vitro, human proximal renal tubular epithelial (HK-2) cells were treated with normal salt or high salt (40 mM NaCl) for 24 hours, receiving recombinant renalase (25ug/mL) or vehicle. In vivo, Dahl salt-sensitive (DS) rats and renalase knockout mice were placed on normal (0.8% NaCl) or high salt (8% NaCl) diet with or without renalase (1 mg/kg) for 4 weeks. Results: In vitro, renalase decreased the phosphorylation levels of protein kinase B (Akt-1), leading to a reduction in the mRNA expression of pro-inflammatory cytokines and oxidative stress induced by high salt. And when knockdown the channel plasma membrane calcium ATPase (PMCA4b) in HK-2 cells, the inflammation and oxidative stress levels induced by high-salt decreased. In DS rats, high salt diet significantly increased the blood pressure and the 24 h urinary sodium excretion. Furthermore, renal inflammation and fibrosis of high salt group were more severe compared to normal salt diet group. Administration of recombinant human renalase reduced kidney inflammation through inhibition of Akt-1 pathway. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals with high salt diet. Treatment with recombinant renalase significantly decreased the kidney inflammation through inhibition Akt-1 expression. Conclusions: Our study provides the first evidence that renalase can ameliorate kidney injury and inflammation through inhibition of the Akt-1 pathway. These results suggest that renalase has potential renoprotective effects in renal injury and inflammation and may be an effective agent for slowing kidney damage.

REVERSAL OF UNILATERAL URETERAL OBSTRUCTION LEADS TO SALT‐SENSITIVE HYPERTENSION

Hypertension is a major cardiovascular disease and 50% of the hypertensive population has salt‐sensitive hypertension. There is mounting evidence that a long‐term consequence of acute kidney injury is increased risk for salt‐sensitive hypertension and chronic kidney disease (CKD). Animal models to study the pathophysiological basis for salt‐sensitive hypertension that follows occurrence of kidney injury are lacking. In the current study, we used a reversal unilateral ureteral obstruction (RUUO) model to study long‐term kidney injury consequences to cause salt‐sensitive hypertension. In this model, we removed the ureteral obstruction 10 days following UUO surgery in 8–10 week old male C57BL/6J mice and divided them into four groups (n=5–6/group): Sham‐Normal Salt Diet, Sham‐8% NaCl Salt Diet, RUUO‐Normal Salt Diet, and RUUO‐8% NaCl Diet. We removed the clip form the ureter day10 day which is a time point the UUO mice demonstrate marked renal fibrosis, inflammation, tubular injury, and lower vascular density. At day 10, the mice were fed a normal or 8% NaCl diet for 4 weeks. Weekly blood pressure was measured till the end of the protocol followed by terminal urine and kidney tissue collection for biochemical, histological, immunohistological, and gene expression studies. There was a progressive and steady rise in systolic blood pressure to 130±5 mmHg in RUUO‐8% NaCl diet group compared to other groups that averaged 113–118 mmHg. Interestingly, RUUO‐8% NaCl group had 50% higher albuminuria compared to Sham‐8% NaCl Salt Diet group. We also demonstrated significant increases in renal mRNA expression of adhesion molecules ICAM and VCAM in RUUO‐8% NaCl diet group compared Sham‐8% NaCl Salt Diet. Most importantly, we demonstrated that RUUO‐8% NaCl diet group had 30% lower vascular density in the outer medullary area and 16% lower cortical vascular density compared to RUUO‐8% NaCl diet group. Overall, these findings present a new animal model to study kidney injury to salt‐sensitive hypertension. In addition, development of salt‐sensitive hypertension and kidney injury in RUUO mice is associated with decreased renal vascular density.

A rapid method for measuring serum oxidized albumin in a rat model of proteinuria and hypertension

Oxidative stress is a risk for and cause of various disease, however, measurements of oxidative stress are either time-consuming or non-specific. Here, we established a rapid method of using high performance liquid chromatography (HPLC) to measure serum oxidized albumin in a rat model. We optimized HPLC conditions for rat oxidized albumin. To validate our method, three-week-old male Sprague-Dawley rats were uninephrectomized and treated normal diet, high salt diet or high salt diet with Tempol, a superoxide dismutase (SOD) mimetic. After 4 weeks of treatment, we analyzed serum oxidized albumin. The main findings are listed as below. (i) Our method of oxidized albumin measurement only takes 16 minutes, with an intra-day and inter-day deviation within 1% and a detection limit concentration of 6.4 mg/ml. (ii) Oxidized albumin levels were significantly higher in the high salt diet group than in the normal salt diet group, and this effect was reversed by Tempol. (iii) Oxidized albumin levels also correlated with urinary protein and 8-isoprostane levels. In conclusion, we have established a simple method for evaluating rat serum oxidized albumin using HPLC. Our method is rapid and has an advantage over conventional methods and may be useful for animal models of oxidative stress.

A Modified Goldblatt 2K1C Kidney Arterial Ligation Method and Salt Consumption: Which one is Best for Hypertensive Mouse Model?

The Goldblatt method is one of several methods that can be used in making mouse model of hypertension. The principle of the Goldblatt method itself aims to cause renovascular hypertension by constricting renal blood vessels that results in hypoxic injury and consequently affects the RAAS (Renin-Angiotensin-Aldosterone System). This study aims to determine whether a modification to the Goldblatt method and an additional high salt consumption could be a more effective method in developing a mouse model of hypertension. This study was done on DDY strain mice with 4 weeks of age and 25-30 grams of weight. A simple ligation to constrict renal blood vessel was used as modification from the original Goldblatt 2K1C (two-kidney, one-clip) method. A total of 32 mice were randomized to receive either a modified Goldblatt, modified Goldblatt with salt diet, sham surgery only, or sham with salt diet. Blood pressure was measured at baseline, first week after intervention, and second week after intervention. The data was then analysed by repeated ANOVA method. Hematoxylin and eosin staining was used to analyse histology appearance. This study showed that using a high salt diet only-method generated a hypertensive state faster compared to the modified Goldblatt method, while the modified Goldblatt method produced a steadier increase of blood pressure. Statistically, there was a significant difference of blood pressure between the sham and salt diet group compared to the other three groups at the first week after intervention (p&lt;0,05). The resulting blood pressure from all the methods used in this study was influenced by time. From all four interventions, it is concluded that the modified Goldblatt 2K1C arterial ligation method with an additional high salt consumption had an effect on mice blood pressure. It is prospective to use the salt intervention method for study of hypertension with a short-time period because its acute effect on the rise of diastolic blood pressure was more rapid than the other three groups. On the other hand, the ligation group produced a steadier increase in diastolic blood pressure, therefore might be effective to be used for study of hypertension in a long-time period.

Long-term low salt diet increases blood pressure by activation of the renin-angiotensin and sympathetic nervous systems

ABSTRACTBackground The aim of this study was to investigate the effect of long-term low salt diet on blood pressure and its underlying mechanisms.Methods Male Sprague-Dawley (SD) rats were divided into normal salt diet group (0.4%) and low salt diet group (0.04%). Blood pressure was measured with the non-invasive tail-cuff method. The contractile response of isolated mesenteric arteries was measured using a small vessel myograph. The effects on renal function of the intrarenal arterial infusion of candesartan (10 μg/kg/min), an angiotensin II receptor type 1 (AT1R) antagonist, were also measured. The expressions of renal AT1R and mesenteric arterial α1A, α1B, and α1D adrenergic receptors were quantified by immunoblotting. Plasma levels of angiotensin II were also measured.Results Systolic blood pressure was significantly increased after 8 weeks of low salt diet. There were no obvious differences in the renal structure between the low and normal salt diet groups. However, the plasma angiotensin II levels and renal AT1R expression were higher in low than normal salt diet group. The intrarenal arterial infusion of candesartan increased urine flow and sodium excretion to a greater extent in the low than normal salt diet group. The expressions of α1A and α1D, but not α1B, adrenergic receptors, and phenylephrine-induced contraction were increased in mesenteric arteries from the low salt, relative to the normal salt diet group.Conclusion Activation of the renin-angiotensin and sympathetic nervous systems may be involved in the pathogenesis of long-term low salt diet-induced hypertension.

A8009 The effect of capsaicin on glomerular damage and TGF-β1/Smads pathway in rats induced by high salt

Objectives: To investigate the protective effects of dietary capsaicin on progression of glomerular damage in Wistar rats induced by high salt. Methods: Thirty-six male Wistar rats were randomly divided into three groups: normal diet group[n = 12, regular salt diet (0.5%)], high salt diet group[n = 12, high salt diet (4%)], and high salt diet plus capsaicin group[n = 12, high salt diet (4%) with 0.02% capsaicin inside]. Tail artery blood pressure was determined by intelligent noninvasive blood pressure monitor-mouse instrument every 4 weeks. The urinary microalbumin, 24-hour creatinine were measured by biochemical methods. Glomerular sclerosis was observed by HE staining, and renal fibrosis was observed by Masson staining. In addition, the mRNA expressions of TGF-β1, Smad2, Smad3, Smad7 were evaluated by real-time PCR and the protein expressions of Transient Receptor potential vanilloid 1 (TRPV1), TGF-β1, p-Smad2/3, Smad7 were observed by immunochemistry and quantitated by western-blot analyses in renal cortex. Results: Firstly, comparing with the normal diet group, the blood pressure, the fibrosis index of the glomerular, the urinary microalbumin were increased in high salt group, creatinine clearance rate was decreased, the mRNA expression of TGF-β1, Smad2, Smad3, Smad7 and the protein expression of TGF-β1, p-Smad2/3, Smad7 were much higher in high salt group as well. Secondly, after the intervention of capsaicin, the blood pressure, the fibrosis index of the glomerular and the urinary microalbumin were much lower than the high salt group, all of the gene and protein expression were also significantly reduced by TRPV1 agonist capsaicin. Conclusion: Dietary capsaicin may prevent glomerular damage induced by high salt diet via intervening TGF-β1/S-mads signal pathway.

Diuretic or sodium-restricted diet for obstructive sleep apnea-a randomized trial.

Interventions that decrease leg fluid retention reduce obstructive sleep apnea (OSA) severity in nonrandomized experiments. We aimed to investigate in a randomized trial the effect of interventions that reduce fluid volume on OSA severity. Men diagnosed with severe OSA were randomized to receive daily spironolactone 100 mg + furosemide 20 mg or nutritional counseling to sodium-restricted diet plus placebo pill or placebo pill. All participants underwent home sleep apnea testing at baseline and after 1 week follow-up. The change in apnea-hypopnea index (AHI) was the primary outcome. The study included 54 participants and all were assessed at follow-up. The average baseline value of the AHI was similar among groups and from baseline to follow-up the AHI reduced 14.4 per cent (δ value -7.3 events per hour; 95% confidence interval, -13.8 to -0.9) in the diuretic group, 22.3 per cent (-10.7; 95% CI, -15.6 to -5.7) in the diet group, and 0.8 per cent (0.4; 95% CI, -2.5 to 3.2) in the placebo group (p = .001 for time × group interaction). None of the patients had their AHI returned to normal. The reduction in the total body water was 2.2 ± 2.2 L in the diuretic group (p < .001) and 1.0 ± 1.6 l in the low salt diet group (p = .002). Sleepiness and neck circumference were significantly reduced only in the diet group (p = .007 and p < .001 for the time × group interactions, respectively). Interventions to reduce bodily fluid content in men with severe OSA promoted a limited decrease of apnea frequency. This finding suggests that rostral fluid displacement affects only partially the OSA severity and/or that other factors prevail in determining pharyngeal collapsibility. Sodium-Restricted Diet and Diuretic in the Treatment of Severe Sleep Apnea (DESALT), https://clinicaltrials.gov/ct2/show/NCT01945801 ClinicalTrials.gov number: NCT01945801.

Abstract P228: Chronically Enhanced Fructose Intake Induces Salt Sensitive Hypertension in Mice and Increases NKCC2 Phosphorylation

High fructose consumption is implicated in hypertension and renal damage in humans. The mechanisms for the renal effects of fructose are not clear. In normal rats, a high fructose diet (20%) does not increase blood pressure, but rapidly (one week) induces a salt sensitive increase in blood pressure. NaCl reabsorption by the thick ascending limb (TAL) is mediated by the NKCC2 cotransporter. Higher NKCC2 activity is involved in salt sensitive hypertension, and its phosphorylation is increased by fructose in rats. We hypothesized that high fructose (20% drinking water), would induce NKCC2 phosphorylation and induce salt sensitive hypertension in normal C57/Bl6J mice. To test this, mice were given 20% fructose in drinking water and after two weeks they were treated for 10 more weeks with either fructose alone or fructose plus 4% Na diet. Tail cuff systolic blood pressure (SBP) was measured after 3 weeks of training. Total NKCC2 expression, GAPDH and NKCC2 phosphorylation at Thre96/101 (P-NKCC2) were measured by Western blot in freshly isolated TALs. Baseline SBP was 112 ± 3 mmHg. A transient increase to 125± 4 mmHg ( p &lt;0.05) was observed after 1 week on fructose which later decreased to 119 ± 3 mmHg ( p =0.14) by week 2. At the end of week 2, high salt chow was added. BP remained normal for 3 weeks and then increased to 137 ± 11 mmHg ( p &lt;0.05) by week 4 on high salt, but not in fructose alone mice. After 10 weeks SBP remained elevated in the fructose plus high salt diet group (124 ± 3 vs 110 ± 5 mmHg; p &lt;0.05). Water intake and urine volume was similar between the two high fructose groups. After 10 weeks, fasting glucose was higher in all mice with fructose (fructose + HS: 231± 8, fructose: 218 ± 7, normal diet: 122 ± 4 mg/dL, p &lt;0.001). A fructose diet increased P-NKCC2 by 248 ± 82% (n=5, p &lt;0.01) by one week and by 61 ± 21% ( p &lt;0.05) at week 12. The combination of high-fructose high salt diet increased P-NKCC2 by 226 ± 75% ( p &lt;0.05) at 12 weeks. Transcriptome profiling of isolated mouse TALs showed that 20% fructose (7 days) increased the expression of genes upstream of the SPAK and AMPK pathway. We conclude that 20% fructose in drinking water leads to a salt dependent increase in SBP, which appears to occur after mice develop abnormalities in glucose metabolism.

Effect of dietary salt intake on epithelial Na+ channels (ENaC) in vasopressin magnocellular neurosecretory neurons in the rat supraoptic nucleus.

Key points A growing body of evidence suggests that epithelial Na+ channels (ENaCs) in the brain play a significant role in the regulation of blood pressure; however, the brain structures that mediate the effect are not well understood.Because vasopressin (VP) neurons play a pivotal role in coordinating neuroendocrine and autonomic responses to maintain cardiovascular homeostasis, a basic understanding of the regulation and activity of ENaC in VP neurons is of great interest.We show that high dietary salt intake caused an increase in the expression and activity of ENaC which resulted in the steady state depolarization of VP neurons.The results help us understand one of the mechanisms underlying how dietary salt intake affects the activity of VP neurons via ENaC activity. All three epithelial Na+ channel (ENaC) subunits (α, β and γ) are located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na+ leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β‐ and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaCs, which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs.

PROTECTIVE EFFECT OF ZINC;

Background: Nutrition is an important determinant of bone health. Micronutrients,other than calcium has been paid less attention to-date in the prevention and treatment of bonediseases. Objective: To evaluate the protective effect of zinc on high salt induced gross changesin humerus and femur of rats. Study design: Analytical control randomized trial. Place andduration of study: Islamic International Medical College, Rawalpindi, hosted the research withthe cooperation of National Institute of Health; Islamabad. The study was approved by Ethicalreview committee of Riphah international university before its initiation. It took six months tocomplete the research (Sep 2015-March 2016). Material and methods: Forty five adult femaleSprague Dawley, 10-12 weeks old rats were used in the study. Three groups were made, eachhaving fifteen rats. Control group C (N=15) received laboratory diet without any alteration.Experimental group A (N=15) were served with high salt diet (8%NaCl) whereas experimentalgroup B (N=15) animals were given high salt diet augmented with zinc (50mg/kg/day).Allgroups were given the diet for eight weeks. Animals were weighed at the start and end ofstudy after which they were sacrificed. Left humeri and femora of all rats were obtained. Weightand mid shaft diameters of bones were recorded. The results were compiled after comparisonamongst all the groups. Results: Marked gross changes were witnessed in experimentalgroups. These changes were of greater severity in high salt diet group as compared to the zincsupplemented group in which reverse beneficial effects were noticed. After zinc administration,there was substantial increase in the weight of animals and bones with concurrent increase inmid shaft diameters. Conclusion: Zinc has a Protective role against high salt induced damageon the gross parameters of bones.