| PartⅠPhosphoproteomic Analysis of Renal Cortex in Chronic Renal Failure Rats on Different Dietary Salt IntakeBackgroundIt is reported that the incidence of hypertension in early stage chronic renal failure is about50%, but that in end-stage is up to60-100%, the persistence of high blood pressure will accelerate the progress of chronic kidney disease, creating a vicious cycle, so the positive control of blood pressure in the various stages of chronic renal failure is of great significance. The increase in dietary salt is the most important environmental factor that promote the development of hypertension, and it also has an independent role on blood pressure, such as the left ventricular hypertrophy, atherosclerosis resistance vascular stenosis, renal hypertrophy, renal fibrosis and so on. As early as in1976, the Ylitalo research team found that the presence of salt sensitivity were indeedly in chronic renal failure rats. Since then Koomans, et al also discovered that there are salt sensitivity in patients with chronic renal failure. Although there are so many researches on salt-sensitive in chronic renal failure, but the mechanism on how the salt diet is to increase blood pressure in chronic renal failure is unclearly known. Studies on the mechanism mainly include:abnormal sodium metabolism, enhanced sympathetic activity, endothelial function impairment (NOS), the baroreceptor damage and so on, the above series of mechanisms are mostly related to disorders of protein phosphorylation signaling pathways. There were so many studies in this area, but they are generally limited to the research on a protein or a pathway of chronic renal failure in specific physiological or pathological state not on the study in the overall protein level, so it is difficult to form a unified network signal for the mechanism.Proteomics is a new discipline from the perspective of the overall activities of protein to recognize a regular life activities, focusing research on whole protein levels in cells and including the protein expression, post-translational modifications. Protein-protein interactions, to obtain comprehensive understanding those processes such as the disease, cell metabolism. The post-translational modification about protein mainly includes the phosphorylation, the S-nitrosylated, acetyl methylation and glycosylation, and in the biosphere, protein phosphorylatione is the most common and important, in which the most studies is the reversible process of phosphorylation and dephosphorylation on a protein translation and modification (PTMs) protein, that regulating all life activities including cell proliferation, development, differentiation, cytoskeleton regulation, neural activity, muscle contraction, metabolism and tumorigenesis process. There are so many human diseases such as tumor, diabetes, autoimmune diseases, cardiovascular disease, all caused due to some abnormal phosphorylation. Kidney contains a variety of phosphorylated protein whose phosphorylation modification plays a very role in regulating normal physiological function of kidney, but abnormal phosphorylation can cause diseases, for example abnormal phosphorylation of WNK cause high blood pressure and the imbalance of acid-base and electrolyte. Therefore analysis the relative changes of protein phosphorylation in different physiological and pathological state can further explore the important biomarkers related to disease development, and help to lay the foundation for revealing molecular mechanism.Although proteomics is more and more used to identificate new target proteins in the organization, to reveal the signal transduction mechanisms, few researches study mechanism for salt sensitivity in chronic renal failure rats applying with phosphorylation proteomics technology. The main purpose of our present study is to focus on the expression of difference for phosphorylated proteins caused by different concentrations of dietary salt in chronic renal failure rats through phosphorylation proteomics technology, to clarifies the occurrence and development of chronic renal failure and the mechanism of salt sensitivity formation in chronic renal failure rats.MethodI Preparation of animal model and the collection of the tissue sample1. Experimental animals and rearing conditionsSD male rats (initial weight,150-180g) were obtained from Sourthern Medical University Animal Experiment Center. The treatment protocol for all the animals was approved by the Animal Experiment and Care Committee of the Sourthern Medical University. The rats were housed in a standard experimental animal laboratory with with free access to food and water.2. Preparation of surgical five-sixth nephrectomy (5/6Nx) animal modelIn rats about200g, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised (totally5/6Nx but fully bilateral adrenal reserved). Control animals were sham-operated with only decapsulation of the kidney.3. In weeks2,4,6,8,10after surgery of rats, collecting orbital venous blood respectively, for testing the serum creatinine and blood urea nitrogen levels.4. Groups allocation and different concentrations of dietary salt for two weeks in ratsIn weeks9after the completion of the second stage surgery, chronic renal failure rats were randomly assigned to:(1) Sham with normal salt dietary group (0.4%NaCl)(n=6)(2) CRF with normal salt dietary group (0.4%NaCl)(n=6)(3) CRF with high salt dietary group (4%NaCl)(n=6) After grouping, measuring tail arterial blood pressure (softron BP-98A, Japan) as the baseline blood pressure. At weeks10beginning with different concentrations of dietary salt rat for2weeks.5. Collection and processing of rats blood, urine and kidney tissue specimens1) At the12weeks after opertation, the consecutive24-hour urine samples were collected in metabolic cages after messuring blood pressure of the animals.2) Anesthetized by intraperitoneal injection in rats:intraperitoneal injection of3%sodium pentobarbital and fixed on the mouse stage with supine position.3) Perform laparotomy and collect abdominal aortic blood, then quickly open the chest, the gavage needle puncture from the left ventricle into the aorta, and cut the right atrial appendage, use4℃ice-cold saline (containing heparin20U/ml)200ml quickly rinse, seeing the kidney, liver, lung, skin and muscle quickly paled, the clear colorless liquid from right atrial appendage flow out.4) Rapidly separate the kidneys, placing in a cuture dish containing4℃precooled1×PBS (pH7.4) and cleaning.5) Removal of the kidney capsule and the surrounding fat, weighed, separation of the cortex of the kidney, wrapped in aluminum foil, frozen in liquid nitrogen,-80℃for storage.6. Serum creatinine and blood urea nitrogen detection (clinical biochemistry analyzer)and quantitative for24h urine protein:Coomassie Brilliant Blue-G250assay method.Ⅱ Renal phosphoproteomic analysis (isobaric tags for relative and absolute quantitation)1. Protein extractionAppropriate amount of tissue block samples were obtained in each group,6samples in each group equally mixed, then grounded in liquid nitrogen, after Cracking, obtaining supernatant for protein quantification using the BCA method.2. SDS gel electrophoresisEach group were approximately20ug protein samples, in accordance with the proportion of5:1(v/v)added5X sample buffer for12.5%SDS-PAGE electrophoresis.3. Tryptic digestion and peptide quantitativeEach group were approximately300μg protein samples, using UA buffer (8M Urea,150mM TrisHCl pH8.0) for protein alkylation and adding trypsin digestion, then performing the OD280peptide quantitative.4. Peptide tagEach group was the same amount of peptide (80ug), according to kit:iTRAQ Reagent-4plex Multiplex Kit (AB SCIEX) instructions to mark, repeat mark one time. The labeled peptide solution were mixed, took1/10volume of mixed peptides desalted and then analyzed by mass spectrometry. Take the remaining9/10of the volume of mixed peptides vacuum freeze-dried, freeze-dried titania for enrichment.5. Titanium dioxide enrichmentVacuum freeze-dry the labeled hybrid peptides solution, add1×DHB buffer for reconstitution. TiO2beads was added a solution, oscillation40min, centrifuged, and the supernatant was removed. With stopper beads into the tip head, add washing bufferl washed three times, Add washing Buffer2washed three times. Elution buffer eluted and phosphopeptides were collected, concentrated in vacuo, dissolved in20μL0.1%FAof10μL for mass spectrometry analysis.6. Mass Spectrometry1) Capillary High Performance Liquid Chromatography(HPLC)Samples using nano-flow the of HPLC liquid phase system Easy nLC1000for separation.2) Identified by mass spectrometrySample separated by capillary HPLC with Q-Exactive mass spectrometer (Thermo Finnigan) mass spectrometry analysis.7. Data Analysis1) mass spectral dataMass spectrometry analysis of the raw data for RAW files, and inventory identification and quantitative analysis with software Mascot2.2and the Proteome Discoverer1.3(Thermo Scientific).2) DatabaseThe use of database:ipi.RAT.v3.87.fasta (Date2011-2-27, protein sequence39925).3) Mascot searchMascot software version for inventory was Mascot2.2. Inventory RAW files submitted by the Proteome Discoverer to Mascot server, select the already established database and database search.4) Quantitative analysisThe Proteome Discovererl.3software according to Ion peak intensity values of the peptides reported conducted quantitative analysis. Peptide quantitative results were the ratio of the signal intensity value of the reference samples and other labelled signal intensity value. Protein quantitative results were the the median as identification of peptide quantitative results. Final quantitative results were normalized of each tag ratio median, to eliminate human factors introducing into the experimental sample volume error.5) Phospho RS NoteMascot software searched Mass spectral data, then analysed by Proteome Discovererl.3software of phosphopeptides (the Phospho RS score, PhosphopRS SEQUENCE probability, phospho RSsite probabilities), the Phospho RS score more than50points and Phospho RS site probabilities greater than75%suggested the higher phosphorylation credibility.Ⅲ StatisticsAll data are representative of the experiment results for repeated more than3times in terms of the mean±standard deviation, All statistical analysis were done by the statistical software SPSS13.0. Two samples mean values comparision used the two independent samples t-test;multiple samples mean values with homogeneity of variance were determined by One-way ANOVA, and pairwise comparisons using LSD method; heterogeneity of variance of multiple samples were compared using the Welch method, pairwise comparisons using Dunnett’s T3method. p<0.05was considered statistically significant.Results:1. Establishment of the renal failure rat modelIn the first10weeks of the two-step5/6nephrectomy, measuring body weight, basic blood pressure and collecting blood sampling for Cr, BUN and the24-hour urine protein in both groups. In weeks10, compared with the group Sham, the systolic blood pressure, serum creatinine, blood urea nitrogen, and24-hour urine protein in the CRF group were markedly increasing (p<0.05), but body weight of rats in all groups kept no significant change(p<0.05).2. Effects of different concentrations of dietary salt on blood pressure,24h urinary protein and urinary sodiumComparison with the normal salt in Sham rats, rats given a low-salt or high-salt diet for14days failed to cause the blood pressure,24h urine protein change significantly (p>0.05). In CRF rats, compared with the normal salt, giving the low-salt diet did not cause the blood pressure,24h urinary protein decreased significantly (p>0.05), but a high-salt diet causes blood pressure24h urine protein significantly higher (p<0.05). In both Sham and CRF group, compared with normal salt, low-salt diet can reduce excretion of urinary sodium,but high-salt diet increased excretion of urinary sodium (p<0.05).3. Effects of different concentrations of dietary salt on rat kidney cortex total protein3.1Identication of total protein SPECTRUM2725proteins were identified by Proteomic iTRAQ technology for each group renal cortex, in which there are2695quantitative protein. The frequency distribution of protein quantitative ratio for identification suggested that most protein ratio (NC/NS, HC/NC)were all close to1, symmetrically, in line with the laws of statistical.3.2Frequency distribution of difference protein ratio Protein quantitative information were compared and the significant index calculated. In the normal diet salt, compared with Sham rats (NC/NS), in CRF rats there were318proteins differentially expressed (p<0.05), of which there are164kinds of protein expression increased and154decreased; In the CRF rats, compared with the normal dietary salt (HC/NC), high-salt inCRF rats there were310proteins differentially expressed (p<0.05), of which157kinds of protein expression increased and153decreased.4. Different concentrations of dietary salt on rat renal cortex phosphorylated peptides4.1Identification of Phosphorylated peptidesWe applied the phosphorylation Proteomics iTRAQ technology to identify the kidney cortex for each group, and identified1911phosphorylated peptides, among it there are1810quantitative information phosphorylated peptides,which corresponding to989phosphorylated proteins, the frequency distribution of Phosphorylated peptides quantitative ratio for identification suggested that most protein ratio (NC/NS, HC/NC)were all close to1, symmetrically, in line with the laws of statistical.4.2Distribution of phosphorylated peptides sitesIdentification of the phosphorylation sites for1810quantitative information on phosphorylated peptides, we identified2565phosphorylated sites, and found a site which occurs phosphorylated peptides the most,and followed by two points phosphorylated peptides (1P1151,2P583,3P59,≥4P17).4.3GO classification of total phosphorylated peptidesBy using Panther online database, we conducted functional classification for the phosphorylated protein which were corresponded to quantitative information phosphorylated peptides. In renal cortical phosphorylated proteins identified by mass spectrometry, protein binding and catalytic activity of molecules is the main functional protein. Phosphorylated protein which were corresponded to quantitative information phosphorylated peptides were performed biological process categories. Phosphorylation proteins in renal cortex involved in metabolism, biological regulation, cells Composition, proliferation, differentiation, apoptosis and other biological processes, wherein metabolic is the most, followed by biological regulation and the forwarding process.4.4Effects of dietary salt on renal cortex phosphorylated peptidesComparing with quantitative information phosphorylated peptides and calculating its significant index. In the normal diet salt, compared with Sham rats (NC/NS), in CRF rats there were197phosphorylated peptides differentially expressed (p<0.05), of which there are113kinds of protein expression increased and84decreased; In the CRF rats, compared with the normal dietary salt (HC/NC), high-salt in CRF rats there were169proteins differentially expressed (p<0.05) of which78kinds of protein expression increased and91decreased.a) GO classification of difference phosphorylated proteinsBy using Panther online database, we conducted functional classification for the phosphorylated protein which were corresponded to difference phosphorylated peptides and Ignored unsorted phosphorylated proteins. In normal salt groups, phosphorylated protein’s main function caused by chronic renal failure corresponded by difference phosphopeptides are nucleic acid binding protein and catalytic activity(NC/NS); In CRF rats, phosphorylated protein’s main function caused by high salt corresponded by difference phosphopeptides are also nucleic acid binding protein and catalytic activity(HC/NC); According to biological classification, In normal salt groups, phosphorylated protein’s main function caused by chronic renal failure corresponded by difference phosphopeptides mainly involved in metabolism, biological regulation, cells composition, proliferation, differentiation, apoptosis and other biological processes, wherein metabolic is the most, followed by biological regulation and the forwarding process(NC/NS); In CRF rats, phosphorylated protein ’s main function caused by high salt corresponded by difference phosphopeptides also involved in metabolism, biological regulation, cells composition, proliferation, differentiation, apoptosis and other biological processes, wherein metabolic is the most, followed by biological regulation and the forwarding process(HC/NC). These reflect the occurrence and development of chronic renal failure and salt sensitivity formation is one of complex extensive adjustment modes. b) Signaling pathway analysis for difference phosphorylated proteinsBy DAVID6.7online analysis software, the KEGG pathways involved by149kinds of difference phosphorylated proteins in NC/NS group and127in HC/NC group were analyzed. The results showed, there were in NC/NS group31differentially phosphorylated proteins involved in metabolism of a variety of substances, lesions adhesion, gap junctions, MAPK, ErbB, mTOR, VEGF, Notch, NOD-like receptor and Calcium signaling pathways, whose function involves cell proliferation, cell cycle, cell communication and apoptosis, drug metabolism, aldosterone regulation of sodium reabsorption and other processes. In HC/NC group there were32differentially phosphorylated proteins involved in metabolism of a variety of substances, lesions adhesion, adhesion connection, mTOR, VEGF and Insulin signaling pathways, whose functions involved in cell proliferation, cell cycle, cell communication and apoptosis, drug metabolic and other processes.c) Interaction analysis for difference phosphorylated proteinsBy using String database, difference phosphorylated proteins in both group NC/NS and group HC/NC were further analyzed. Among difference phosphorylated proteins in NC/NS group, there were36proteins neted, that those phosphorylated proteins are likely signaling pathway members which related to the occurrence and development of chronic renal failure. The Polr2a, Srrml and Srrm2were the most connected, which illustrate that these three phosphorylated proteins may be central node of signaling pathway network related to the development and occurrence of chronic renal failure, they are probably important targets of signaling pathways phosphorylation related to the development and occurrence of chronic renal failure. Among difference phosphorylated proteins in HC/NC group, there were24proteins neted, that those phosphorylated proteins are likely signaling pathway members which related to salt sensitivity formation of chronic renal failure. The Lmna, Des, Tnni3, Mydpc3, Vcl, Myh6, Myh7and Myhll were the most connected, which illustrate that these8phosphorylated proteins may be central node of signaling pathway network related to salt sensitivity formation of chronic renal failure, they are probably important targets of signaling pathways phosphorylation related to salt sensitivity formation of chronic renal failure.Conclusion1. Successfully use iTRAQ technique to conduct phosphorylated proteomic analysis on rats kidney cortex, constructed phosphorylated protein spectrum of kidney cortex in chronic renal failure rats; identified1,911kinds of phosphorylated peptides,in which there are1810kinds having quantitative information and corresponding to989kinds of phosphorylated proteins.2. Successfully found149kinds of difference phosphorylated proteins which may related to the occurrence and development of chronic renal failure and and127may be associated with formation of salt sensitivity in chronic renal failure.3. Based on the identification of signaling pathway phosphorylated proteins related to the occurrence and development of chronic renal failure and salt sensitivity formation, we established phosphorylated proteins signaling network related to occurrence and development of chronic renal failure and salt sensitivity formation signaling pathways. Part ⅡEffect of Salt Intake on Renin Angiotensin System of the Brain Nucleus Tractus Solitarii in Chronic Renal Failure RatsBackgroundThe prevention and treatment of chronic kidney disease has become one of the important public health problem in the world, and chronic renal failure is a syndrome of metabolic disorders and clinical symptoms caused by chronic kidney disease which progress to a certain period. Most of the CRF patients suffer from varying degrees of hypertension, which the prevarence rate was about60%to100%, and persistent high blood pressure will accelerate the progress of chronic kidney disease and create a vicious cycle, therefore actively controlling blood pressure well in patients with chronic renal failure has important implications in treatment stages. It is well known that the increase in dietary salt is the most important environmental factors that promote the development of hypertension, As early as in1976, the Ylitalo P research team found that the presence of salt sensitivity were in chronic renal failure rats.Since then the HA Koomans, et al discovered that there are salt sensitivity in patients with chronic renal failure, but the mechanism of this salt sensitivity is not fully clear.The renin-angiotensin system plays a very important role in the formation and development of hypertension in chronic renal failure, in addition to the systemic RAS involving in the process, brain RAS is also involved in the development of hypertension. Teruya et al in1995confirmed that salt-sensitive is related to brain angiotensin, subsequently Leenen research team also found that high salt can act on the brain renin-angiotensin system in Dahl salt-sensitive rats and salt intake positively correlats with sympathetic activity. Sympathetic overactivity and the deterioration of the blood pressure caused by the high-salt diet in spontaneously hypertensive rats (SHR), can be blocked by AT1receptor blocker losartan through intraventricular injection; Dahl S rats with high salt diet, losartan through intraventricular injection prevents the excessive activity of sympathetic nervous system, and inhibits the increase of blood pressure. It is clear that central RAS is closely related to the formation of salt sensitivity, but so far the exact mechanism is unknown.There are all RAS components in the brain, in which the brain RAS system especially the region of the brain stem cardiovascular regulation system (NTS, RVLM) plays an important role in the regulation of blood pressure by regulating sympathetic activity. In these regions, NTS which is the initiation site of tongue pharyngeal nerve terminal in vagus nucleus, is an important area of regulating baroreceptor reflex and for the set-point of arterial blood pressure. Research has shown that microinjection of ANGII into NTS can weaken baroreceptor sensitivity and cause high blood pressure. Increasing ANGII in blood can reduce the sensitivity of the baroreceptor through the blood-brain barrier acting on NTS, and intracerebral injection of AT1receptor blockers can be completely removed its role. Thereby we can infer that the RAS in NTS plays an important role in the regulation of blood pressure.In this study, through the intervention of different concentrations of salt in chronic renal failure rats, we observed the RAS change of brain NTS, with chronic renal failure rats as a model, and explored the formation mechanism of salt sensitivity in rats with chronic renal failure by intracerebral injection of chlorine with high salt diet.MethodI Preparation of Animal Model and the collection of the tissue sample1. Preparation of rats5/6nephrectomy model for chronic renal failure and salt stimulationChosing body weight about200g rats operated2/3left kidney nephrectomy, after one week,full right kidney nephrectomy conducted (but bilateral adrenal gland fully reserved), and sham-operated rats only peeled renal capsule. In weeks2,4,6,8,10after surgery, orbital venous blood was collected respectively, testing serum creatinine andblood urea nitrogen levels in rats. In weeks9after the completion of the second stage surgery, sham-operated and part of chronic renal failure rats were randomly assigned to:After grouping, measuring tail arterial blood pressure (softron BP-98A, Japan) as the baseline blood pressure. At weeks10beginning with different concentrations of dietary salt rat for2weeks.2. Intraventricular injection of losartan potassim in rats with chronic renal failure during high salt dietIn weeks9after the completion of the second stage surgery, chronic renal failure rats were randomly assigned to:(1) CRF with high salt dietary group (n=15)(2) CRF with high salt dietary and intraventricular injection of artificial cerebrospinal fluid group (I.C.V CSF,12μl/24h)(n=15)(3) CRF with high salt dietary and intraventricular injection of losartan potassium group (I.C.V losarta,1mg/kg BW.d,12μl/24h)(n=15)After grouping, measuring tail arterial blood pressure (softron BP-98A, Japan) as the baseline blood pressure.ICV infusion:The third ventricle of rats was located by the rat stereotaxic apparatus, an osmotic mini-pump (model2002, Alzet Corp.,CA) containing drug or CSF was implanted subcutaneously at the back of the neck and connected to the free end of the cannula. At weeks10beginning with different concentrations of dietary salt rat for2weeks.3. Collection and processing of rats blood, urine and kidney tissue specimens1) At the12weeks after opertation, the consecutive24-hour urine samples were collected in metabolic cages after messuring blood pressure of the animals.2) Anesthetized by intraperitoneal injection in rats:intraperitoneal injection of3%sodium pentobarbital and fixed on the mouse stage with supine position.3) Perform laparotomy and collect abdominal aortic blood, then quickly open the chest, the gavage needle puncture from the left ventricle into the aorta, and cut the right atrial appendage, use4℃ice-cold saline (containing heparin20U/ml)200ml quickly rinse, seeing the kidney, liver, lung, skin and muscle quickly paled, the clear colorless liquid from right atrial appendage flow out.4) Each group rats (n=5)4℃used precooled normal saline for rapid infusion, then for4℃4%paraformaldehyde solution (pH7.2)400ml perfusion1.5h, then carefully cut the skull and brain, in rats brain mold separating the solitary tract nucleus, fixed at4%paraformaldehyde, for used to the brain tissue in paraffin sections immunohi stochemistry. 5) Each group of the remaining mice (n=5) After rapid infusion of4℃ice-cold saline, opened the skull and got the brain, isolated solitary tract nucleus in the rat brain mold, wrapped in aluminum foil, frozen in liquid nitrogen-80℃for storage.Ⅱ Serum creatinine, urea nitrogen, Sodium, urinary sodium and24h urinary protein were detectedⅢ Determination of AGT, ACE and AT1mRNA by real-time PCRPut the rats solitary tract nuclear tissues in1.5ml EP tubes, according to tissue weight:the TRIZOL=the ratio of1:10repeated pipetting, extracted tissue RNA, reversed transcription, qRT-PCR detection of AGT, ACE, AT1mRNA expression levels.IV Determination of AGT, ACE and AT1protein expression by immunohistochemistryV Determination of ACE and AT1R protein expression by Western BlottingRemoving the tissue stored at-80℃, weighting, according to tissue weight: Lysates=1:4proportion adding in Lysates, repeated pipetting with a5ml syringe, the above operations were carried out on ice, after the completion of the homogenate,13,000g,4℃centrifugal for40min, removing the lipid droplets floating in the upper, extracting homogenates of the intermediate layer, the total protein of the nuclear organization was the solitary tract, adding the SDS (5x) sample buffer,95℃boiling protein, Western Blotting detecting ACE and AT1R protein expression levels in solitary tract nuclear tissue.VI Statistical methodsAll data are representative of the experiment results for repeated more than3times in terms of the mean±standard deviation, All statistical analysis were done by the statistical software SPSS13.0. Comparison with the constant term, one-sample t-test, Two samples mean values comparision used the two independent samples t-test;multiple samples mean values with homogeneity of variance were determined by One-way ANOVA, and pairwise comparisons using LSD method; heterogeneity of variance of multiple samples were compared using the Welch method, pairwise comparisons using Dunnett’s T3method. p<0.05was considered statistically significant.Results1. Establishment of the renal failure rat modelIn the first10weeks of the two-step5/6nephrectomy, measuring body weight, basic blood pressure and collecting blood sampling for serum creatinine (Cr,blood urea nitrogen (BUN) and the24-hour urine protein in both groups. In weeks10, Compared with the group Sham, the systolic blood pressure, serum creatinine, blood urea nitrogen, and24-hour urine protein in the CRF group were markedly increasing (p<0.05), but the weight kept no marked changein all groups (p<0.05).2. Effects of different concentrations of dietary salt on blood pressure in ratsComparison with the normal salt in Sham rats,rats given a low-salt or high-salt diet for14days failed to cause the blood pressure change significantly (p>0.05). In CRF rats, compared with the normal salt, giving the low-salt diet did not cause the blood pressure decreased significantly (p>0.05), but a high-salt diet causes blood pressure significantly higher (p<0.05).3. Effects of different concentrations of dietary salt on24-hour urine protein in ratsComparison with the normal salt in Sham rats,rats given a low-salt or high-salt diet for14days failed to cause the24-hour urine protein change significantly (p>0.05). In CRF rats, compared with the normal salt, giving the low-salt diet did not cause the24-hour urine protein decreased significantly (p>0.05), but a high-salt diet causes24-hour urine protein significantly higher (p<0.05).4. Effects of different concentrations of dietary salt on body weight, serum sodium and urinary sodium in ratsIn both Sham and CRF group, compared with normal salt, rats given a low-salt or high-salt diet for14days failed to change body weight; low-salt diet can reduce excretion of urinary sodium, and high-salt diet can increase excretion of urinary sodium (p<0.05); low-salt or high salt diet had no effect on serum sodium in Sham group (p>0.05), but high-salt diet could increase serum sodium levels in CRF group (p<0.05).5. Effects of different concentrations of dietary salt on RAS of Nucleus Tractus Solitarii in rat brain5.1The mRNA expression of RAS in Nucleus Tractus Solitarii under Different concentrations of dietary salt in ratsFor the normal salt diet, in the CRF group, the AGT’s and ACE’s mRNA expression of RAS in Nucleus Tractus Solitarii in rat brain increased significantly compared with Sham group (p<0.05); In low salt diet, for the CRF group, the AGT’s and AT1mRNA expression of RAS in Nucleus Tractus Solitarii in rat brain increased significantly compared with Sham group (p<0.05); In high salt diet, for the CRF group, the ACE’s and AT1mRNA expression of RAS in Nucleus Tractus Solitarii in rat brain increased significantly compared with Sham group (p<0.05); whether Sham group or CRF group, high-salt diet increases expression of the AGT’s, ACE’s and AT1mRNA of RAS in Nucleus Tractus Solitarii in rat brain compared with normal salt (p<0.05).5.2Protein expression of RAS in Nucleus Tractus Solitarii under Different concentrations of dietary saltWhether in low salt, normal salt or high salt diet, for the CRF group, expression of AGT, ACE,ANGII and AT1of RAS in Nucleus Tractus Solitarii in rat brain increased significantly compared with Sham group (p<0.05); in Sham group, low-salt diet inhibits expression of AGT, ACE, ANGII and AT1of RAS in Nucleus Tractus Solitarii i... |
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