Dissertation
Dissertation > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Renal failure

Effect of Salt Intake on Heart Phosphoproteome and RAS of OVLT in CRF Rats

Author SuZhengXiu
Tutor HouFanFan
School Southern Medical University,
Course Internal Medicine
Keywords Chronic renal failure Hypertension Phosphoproteomics Heart SaltintakeChronic Renal Failure (CRF) Organum Vasculosum of the LaminaTerminalis (OVLT) Salt diet Renin-Angiotensin-System (RAS)
CLC R692.5
Type Master's thesis
Year 2013
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Part Ⅰ:Phosphoproteomics study of the hearts in a chronic renal failure rat model with high salt intakeBackgroundChronic Kidney Disease (CKD) is a global public health problem. Cardiovascular disease (CVD), which is the most important reason for death in patients with chronic renal failure, is the main factor affecting the prognosis of chronic kidney disease, and the mortality of CVD is47%in patients with dialysis.The spectrum of cardiovascular disease (CVD) in patients with chronic renal insufficiency includes left ventricular hypertrophy (LVH) and dilatation, ischemic heart disease, and peripheral vascular disease. End-stage renal disease (ESRD) is always associated with cardiovascular disease, however, CVD is not found only in patients with ESRD. In fact, the prevalence of LVH starts to increase in early CKD, and to become almost universal in patients with ESRD. Moreover, Left ventricular hypertrophy (LVH) is a validated marker of cardiovascular end-organ damage. Indeed, renal impairment in isolation may be directly responsible for poor outcomes and has a causal role in the progression of left ventricular systolic dysfunction, retention of sodium and fluid, increase in cardiac filling pressures and subsequent ventricular dilatation, through continued activation of neurohumoral pathways. Prolonged and inappropriate activation of both the sympathetic nervous system (SNS) and the renin-angiotensin system occurs in many forms of renal diseases and is well established as playing a key role in the pathophysiology of heart failure. Related conditions such as diabetes, obesity, hypertension, as well as the presence of renal dysfunction per se lead to oxidative stress, low-grade inflammation, dyslipidaemia, elevated circulating pro-inflammatory cytokines, which may all play a role in the association of renal and cardiovascular disease. Meanwhile, CVD can also directly act on the kidneys to promote the occurrence and development of chronic kidney disease by reducing renal perfusion and accelerate renal arteriosclerosis.Hypertension is highly prevalent in patients with chronic kidney disease (CKD), as defined by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative. As either the cause or the consequence of CKD, hypertension is an important independent factor determining the rate of loss of renal function. Hypertension is also a significant independent risk factor for cardiovascular events in patients with CKD, the leading cause of their morbidity and mortality. There is overwhelming evidence from epidemiological, intervention and genetic studies in humans and animals that indicate a strong dependence of the blood pressure on the salt intake, Salt-sensitive individuals have a greater rate of cardiovascular complications than salt-resistant individuals independently from classic cardiovascular risk factors.Prolonged dietary salt-loading promoted structural and function derangements of the target organs of hypertensive disease independent of its pressor action. With respect to the experimental laboratory cardiac responses to salt overload, there was a small but significant increase in arterial pressure; In addition, there was a more profound increase in ventricular fibrosis, bilateral ventricular ischemia and impaired diastolic function. However, Koomans et al found that lower sodium intake markedly diminished BP in patients with advanced CKD. Dietary sodium restriction will prevent cardiovascular and renal outcomes through structural and functional pathophysiologic changes induced by sodium that are independent of the RAS, and will further diminish that risk by reversing those structural and functional derangements. The trial of hypertension prevention studies (TOPH I and II), involving2,382prehypertensive participants, reported that dietary sodium restriction (which had been previously shown to lower blood pressure) demonstrated highly significant cardiovascular risk reduction (by) than those individuals not receiving such a diet. A growing body of evidence suggests that salt intake reduction confers a risk reduction effect on cardiovascular disease end points.Proteomics is used to study the complexity of proteins, their roles, and biological functions. It is based on the premise that the diversity of proteins, comprising their isoforms, and post translational modifications (PTMs) underlies biology. Many key cardiac functions such as calcium homeostasis, cardiac contraction, and cell survival signaling are regulated via protein phosphorylation/dephosphorylation. Disruptions of phosphorylation signaling have been linked to several cardiovascular diseases such as ischemia/reperfusion (I/R) injury and cardiac hypertrophy, and thus protein phosphorylation signaling modulators have emerged as critical targets of cardiovascular drugs. Cardioproteomics (Cardiovascular proteomics) is fast becoming an indispensible technique in deciphering changes in signaling pathways that occur in cardiovascular diseases (CVDs).Although the importance of cardiovascular complications in chronic kidney disease is widely recognized, and there are many studies on pathogenesis of the both diseases, Proteomics approach to study the potential mechanisms of CVD in Chronic renal failure is rare. Therefore, we are trying to take advantage of phosphoproteomics to observe the protein expression profile changes of heart in chronic renal failure rats, and hope that it will be able to provide clues for future research work and clinical treatment.Methods1. Preparation of a chronic renal failure rat model and the collection of the tissue sample1.1Experimental animals and rearing conditionsSD male rats (initial weight,150-180g) were obtained from Animal Experiment Center of Southern Medical University. The treatment protocol for all the animals was approved by the Animal Experiment and Care Committee of the Southern Medical University. The rats were housed in a standard experimental animal laboratory with12:12-h light-dark schedule and room temperature of22℃, with free access to food and water.1.2Preparation of animal model and subsequent processingThe animals were divided into two groups, CRF group (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thirds of the left kidney) and sham operation group (control). Nine weeks after the operation, the CRF rats were randomized into three subgroups:1) Sham rats+Normal salt diet (0.4%NaCl,NS,n=6)2) CRF rats+Normal salt diet (0.4%NaCl, NC, n=6)3) CRF rats+High salt diet (4%NaCl,HC,n=6)The baseline blood pressure (systolic blood pressure) of conscious animals was indirectly measured by the tail-cuff method. The rats were kept on different salt diet for14days.1.3Experimental data and Specimen collection1.3.1Blood pressure and urine collectionThree days before the end of stimulus,24-hour urine samples were collected for three consecutive days, and the blood pressure was indirectly measured by the tail-cuff method also.1.3.2Blood collection and specimen collectionRats were anesthetized with3%pentobarbital sodium (35mg/kg, i.p.). Abdominal aortic blood was collected into chilled vacuum tubes, and serum and plasma samples were separated and stored at-80℃until assayed. To collect heart tissue for follow-up study, rats were perfused with200ml of ice-cold normal saline. The whole heart of some were harvested following thoracotomy and washed with0.1M PBS. The harvested heart were then weighed, and after removing the atria and right ventricle, the free wall of left ventricle were quickly placed in liquid nitrogen and kept in the-80℃refrigerator until protein extraction.1.3.3Serum creatinine was detected by clinical automatic biochemical analyzer. 1.4StatisticsAll values were presented as mean±SE. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical software SPSS13.0.The accepted level of significance was P<0.05.2. Cardiac phosphoproteomic analysis2.1Protein extractionThe frozen heart tissues were mixed with an equal amount of the six samples within the group and pulverized in liquid nitrogen, Bicinchoninic acid was used to determine the protein concentration after completely lysis.2.2SDS gel electrophoresisApproximately20μg protein sample was taken from each group,5X loading buffer was added in accordance with the ratio of4:1(v/v) and12.5%SDS gel electrophoresis was utilized.2.3Tryptic digestion and peptide extractionApproximately300μg protein sample was taken from each group, the protein was digested according to the Laboratory’s protocol, Bradford method was used to determine the peptide concentration after completely digestion.2.4iTRAQ labeling of cleaved peptidesApproximately8Oμg peptide sample was taken from each group, and then labeled with iTRAQ Reagent-4plex Multiplex Kit (AB SCIEX) following the manufacturer’s instructions. After labeling with iTRAQ, the six samples were combined and subjected to Titanium dioxide enrichment.2.5Titanium dioxide enrichmentThe mixed peptide solution was desalted with1xDHB buffer after Vacuum freeze-drying, and enriched for phosphopeptide following the Laboratory’s protocol. Enriched peptides were desalted again prior to LC-MS/MS.3. Mass Spectrometry3.1High Performance Liquid Chromatography (HPLC)Nano-flow HPLC system Easy nLC1000was used for the separation of the sample.3.2Mass spectrometry analysisQ-Exactive Mass spectrometer (Thermo Finnigan) was used for mass spectrometry analysis after HPLC.4. Data Analysis4.1Mass spectrometry dataRAW file was the original data of the mass spectrometry, Mascot2.2Software and Proteome Discovererl.3(Thermo Scientific) were used to investigate the database for qualitative and quantitative analysis.4.2Databaseipi.RAT.v3.87.fasta (Date2011-2-27,39,925protein sequences)4.3Mascot searchThe Mascot software version of Mascot2.2was selected. RAW files were submitted by Proteome Discoverer to Mascot server, and a good database was selected for Database search.4.4Quantitative analysisAccording to the report of the peptide fragment ion peak intensity value, the Proteome Discoverer1.3software was used for quantitative analysis. The quantitative result of the peptide was the ratio of the signal intensity value of the reference sample label to the signal strength values of other labels. The median of the quantitative results of the peptide is the quantitative result of the peptide. The final quantitative result was normalized to the median ratio of each label, in order to eliminate the pipetting error originated from the operator in the experiment.4.5Phospho RS NotePhosphorylated peptides were analyzed by Proteome Discovererl.3software (Phospho RS score, Phosphop RS sequence probability, Phospho RS site probabilities) after Mascot software search, Phospho RS score more than50and the value of Phosphop RS sequence probability more than75%represented high phosphorylation credibility. Results1. General data1.1Blood biochemical results before Stimulation10weeks after Modeling, The rats were weighed and the blood pressure was indirectly measured by the tail-cuff method. Then we collected the blood of rats for serum creatinine measurement. We found that serum creatinine, arterial blood pressure and24-hour urine protein of the CRF rats were significantly higher and no body weight change compared with the sham group (Independent samples T-test, BW, t=1.559, P=0.14; Scr, t=-4.021, P=0.01; SBP, t=-2.597, P=0.02;24hU Protein, t=-6.686,P=0.01)1.2Blood biochemical results after StimulationHigh salt diet for14days, compared with sham rats, Heart weight, heart weight/body weight ratio, arterial blood pressure,24-hour urine protein excretion of CRF rat were significantly increased, and can be exacerbated by high salt intake, In addition, serum sodium and the24h urinary sodium excretion were increased in CRF rats with high salt intake (One-Way ANOVA, Heart weight, F=25.96, P=0.001; Heart weight/Body weight ratio, F=49.960,P=0.000; SBP, F=13.635,P=0.001; S Na+, F=7.683,P=0.004;24-hour urinary sodium excretion, F=8.901, P=0.000;24-hour urinary protein excretion, F=64.782, P=0.000)2. Mass spectrum analysis results2.1Quantitative results of total protein before TiO2enrichment1877proteins were identified in this experiment, of which1830were quantitatively.226proteins were changed in the normal-salt CRF group compared with the normal-salt sham group, of which107increased,119decreased;220proteins were changed in the high-salt CRF group compared with the normal-salt CRF group, of which120increased,100decreased.2.2Quantitative results of phosphopeptides after TiO2enrichment1884phosphorylated peptides were identified in this experiment, of which1724were quantitatively.165phosphorylated peptides were changed in the normal-salt CRF group compared with the normal-salt sham group, of which89increased,76decreased;172phosphorylated peptides were changed in the high-salt CRF group compared with the normal-salt CRF group, of which83increased,89decreased.2.2.1Gene ontologyTo understand the functional distribution of the763unique phosphoproteins identified from hearts, DAVID6.7were used to enrich the overrepresented gene ontology (GO), GOMF analysis rev ealed a n ove rrepresen ta tion of kinase activity and binding activity, the latter includes protein, nucleotide, and nucleic acid binding. GOBP analysis indicated that proteins related to protein metabolism and modification, nucleic acid metabolism, response to different stimuli, growth and development, transport, and cellular component organization were overrepresented.2.2.2Analysis of phosphorylation sitesTo precisely assign the phosphorylation sites within a peptide, we used PTM score to calculate the probabilities of phosphorylation at each site. We could localize723phosphosites with high confidence as class I phosphorylation site (P>0.75). Around58.1%of the phosphopeptides identified were found to be singly phos-phoryl ated, and other f ract ions were either doubly (36.4%), triply (4.8%), or more highly (0.6%) phosphorylated. We then analyzed the distribution of the identified class Ⅰ phosphorylation sites, the observed results were14phosphotyrosine sites (1.4%),52phosphothreonine sites (5.2%), and565phosphoserine sites (56.4%).2.2.3Protein interaction analysisa STRING analysis revealed a protein association network, in which15phosphorylated proteins (Des、Vc1、Gja1、Tnni3、Mybpc3、Myh6、Lmna、Myoz3、 Tns1、Pxn、Ctnna1、Pkp2、Dsp、Map1b、Dyncllil) in NC/NS group and23phosphorylated proteins (Des、Vc1、Gja1、Tnni3、Mybpc3、Myh6、Myh7、Myh11、 Myh9、Ryr2、Jph2、Lmna、Vim、Myoz3、Srrm1、Srrm2、Hnrpd、Map1a、Map1b、 Khdrbs2、Hsd17b8、Eif4b、Eif3s9) in HC/NC group can be networked.2.2.4Signaling pathway analysis To reveal the potential signaling pathways represented by the heart phosphoproteome, we searched the identified phosphoproteins against the rat KEGG pathway database. many fundamental biological pathways were highlighted by phosphoproteins identified in this study, including calcium signal transduction pathway, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, the heart muscle contraction, actin cytoskeleton regulation, aldosterone regulation of sodium reabsorption, insulin signaling pathway, MAPK, sugarglycolysis/gluconeogenesis, VEGF and gap junctions in NC/NS group and calcium signal transduction pathway, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, the heart muscle contraction, actin cytoskeleton regulation, aldosterone regulation of sodium reabsorption, insulin signaling pathway, MAPK, sugarglycolysis/gluconeogenesis, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, mTOR signaling pathway and gap junction in HC/NC group.Conclusion1. Phosphorylation level of140protein of the heart in chronic renal failure had changed, in which15phosphorylated proteins in NC/NS group can form a network. many fundamental biological pathways were highlighted by phosphoproteins identified in NC/NS group, including calcium signal transduction pathway, dilated cardiomyopathy and hypertrophic cardiomyopathy;2. Phosphorylation level of138protein of the heart in chronic renal failure with high salt intake had changed,23phosphorylated proteins in HC/NC group can form a network. many fundamental biological pathways were highlighted by phosphoproteins identified in HC/NC group, including calcium signal transduction pathway, dilated cardiomyopathy and hypertrophic cardiomyopathy. Part II:Effect of different salt intake and intraventricular injection of losartan on RAS of the OVLT in rats with chronic renal failureBackgroundThe organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and area postrema (AP) belong to a group of structures known as the circumventricular organs (CVOs). Located in the walls of the ventricular system of the brain, these CVOs are highly vascularized structures lacking the normal blood-brain barrier (BBB). More detailed studies of rats, sheep and dogs in which the OVLT or anteroventral wall of the third ventricle had been ablated showed that water drinking and vasopressin secretion in response to acute increases in plasma osmolality were severely inhibited by these lesions, and it was proposed that the OVLT could be the site of osmoreceptors subserving thirst and vasopressin secretion. There is an association between OVLT and other nucleus such as SFO, MnPo, PVN, and SON, which all play an important role in regulation of fluid balance and cardiovascular function.The renin-angiotensin system (RAS) plays a pathophysiologic role in the progression of chronic renal disease. Inhibition of angiotensin II (Ang II) in experimental and human renal diseases decreases proteinuria, reduces histologic evidence of renal injury, and slows progression to end-stage renal failure. The renin-angiotensin system (RAS) is a hormonal system that controls body fluid volume, blood pressure, and cardiovascular function in both health and disease states.Hyperactivity of the brain RAS has been confirmed to be associated with the development of hypertension in spontaneously hypertensive and salt-sensitive hypertensive animal model. All components of the RAS system are discovered in the brain, including prorenin, ACE, ANGII and specific AT1receptor. Moreover, AT1receptor is found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. In addition, studies have shown that intraventricular injection of losartan can reverse the hypertensive effect induced by intravenous ANGII, which indicated central nervous system may be involved in the development of hypertension.Renal failure patients with hypertension show evidence of an abnormal relationship between sodium and the renin-angiotensin system in that circulating level of renin and angiotensin II is abnormally high in relation to exchangeable sodium. The abnormality may well contribute to the hypertension in this syndrome. An excess of dietary salt is the most common environmental factor that contributes to the pathogenesis of hypertension. Salt restriction, which helps control of hypertension in patients with CRF, can enhance Effect of antihypertensive drugs, and delay the progression of chronic kidney disease. However, the high sodium intake can attenuate Effect of antihypertensive drugs, especially in weakening the antihypertensive effect of ACEI and ARB, which may be caused by the water and sodium retention, activation of the sympathetic nervous system and the RAS system activation.In our study, we intends to select OVLT, an area lacking the normal blood-brain barrier and being related with the regulation of water and electrolyte balance and cardiovascular function, and give different salt diet or intraventricular injection of losartan to stimulate rats with chronic renal failure, in order to confirm whether the RAS system of OVLT is involved in the mechanism of hypertension in rats with chronic renal failure.MethodEffect of different salt intake on RAS of the OVLT in rats with chronic renal failure1. Preparation of a chronic renal failure rat model and the collection of the tissue sample1.1Experimental animals and rearing conditionsSD male rats (initial weight,150-180g) were obtained from Animal Experiment Center of Southern Medical University. The treatment protocol for all the animals was approved by the Animal Experiment and Care Committee of the Southern Medical University. The rats were housed in a standard experimental animal laboratory with 12:12-h light-dark schedule and room temperature of22℃, with free access to food and water.1.2Preparation of animal model and subsequent processingThe animals were divided into two groups, CRF group (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thirds of the left kidney) and sham operation group (control). Nine weeks after the operation, the CRF rats were randomized into three subgroups:The baseline blood pressure (systolic blood pressure) of conscious animals was indirectly measured by the tail-cuff method. The rats were kept on different salt diet for14days.1.3Experimental data and Specimen collection1.3.1Blood pressure and urine collectionThree days before the end of stimulus,24-hour urine samples were collected for three consecutive days, and the blood pressure was indirectly measured by the tail-cuff method also.1.3.2Blood collection and specimen collectionRats were anesthetized with3%pentobarbital sodium (35mg/kg, i.p.). Abdominal aortic blood was collected into chilled vacuum tubes, and serum and plasma samples were separated and stored at-80℃until assayed. To collect brain tissue for follow-up study, rats were perfused with200ml of ice-cold normal saline. The tissue of OVLT was isolated from the brain and quickly placed in liquid nitrogen and then kept in the-80℃refrigerator until protein and total RNA extraction.The remaining rats were continued perfused with4%400ml paraformaldehyde, After completion, the brain was removed and cut into chunks according to the respective requirements, fixed for6hours, then dehydrated in a graded with different concentrations of alcohol, Finally, the samples were embedded in paraffin and sliced in5μm sheet.1.3.3Serum creatinine was detected by clinical automatic biochemical analyzer.1.3.4Serum electrolytes were detected by clinical automatic biochemical analyzer.1.3.524h urinary protein was detected by Bradford method.2. Determination of RAS protein expression2.1Western blottingThe tissue of OVLT was taken out from the-80℃refrigerator and transferred to1.5ml EP tubes, the lysate was added equal to the ratio of1:4to tissue weight, a5ml Syringe was used to repeatedly pipetting samples on ice. After complete lysis the lysate was incubated on ice for15min and then centrifuged at13,000xg for40min at4℃. The supernatant was used to measure protein Concentration by the Bradford method. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to95℃and then AT1receptor and ACE proteins were detected by western blotting.2.2ImmunohistochemistryTake three paraffin sections of OVLT in each rat, after dewaxing and Graded ethanol hydration, immunohistochemical staining was adopted, photographs were captured under the microscope by200X and analyzed using Image-Pro Plus software, Finally, the number of positive cells were counted.3. Determination of RAS mRNA by real-time PCRTotal RNA of the tissue of OVLT was extracted with TRIZOL reagent. AT1receptor and ACE mRNA were detected by real-time PCR according to the manufacturer’s protocol.Effect of intraventricular injection of losartan on RAS of the OVLT in a chronic renal failure rat model with high salt intake4. Preparation of a chronic renal failure rat model and collection of the tissue sample. 4.1Experimental animals and rearing conditionsSD male rats (initial weight,150-180g) were obtained from Animal Experiment Center of Southern Medical University. The treatment protocol for all the animals was approved by the Animal Experiment and Care Committee of the Southern Medical University. The rats were housed in a standard experimental animal laboratory with12:12-h light-dark schedule and room temperature of22℃, with free access to food and water.4.2Preparation of animal model and subsequent processingAll animal procedures were approved by the Animal Experiment and Care Committee of the Southern Medical University. Male Sprague-Dawley rats (initial weight,150-180g, purchased from Southern Medical University Animal Experiment Center) were maintained under standardized conditions. The animals were subjected to5/6nephrectomy (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thirds of the left kidney). Nine weeks after the operation, the CRF rats were randomized into three subgroups.(1) CRF+high salt diet group (HC, n=15)(2) CRF+high salt diet+icv-CSF group (12μl/h, HC+icv-CSF, n=15)(3) CRF+high salt diet+icv-losartan group (lmg/kg/day,12μl/h, HC+icv-los, n=15)The base line blood pressure (systolic blood pressure) was determined in conscious rats by the indirect tail-cuff method.ICV infusion:The third ventricle of rats was located by the rat stereotaxic apparatus, an osmotic mini-pump (Model No.2002, 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. The rats received high salt diet (4%NaCl) for14days.4.3Experimental data and specimen collection4.3.1Blood pressure and urine collection The Same as above4.3.2Blood collection and specimen collection The Same as above 4.3.3Serum creatinine and urea nitrogen detection The Same as above4.3.4Serum electrolytes detection The Same as above4.3.524h urinary protein The Same as above5. Determination of RAS protein expression The Same as above6. Determination of RAS mRNA by real-time PCR The Same as above7. StatisticsAll values were presented as mean±SE. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical software SPSS13.0.The accepted level of significance was P<0.05.ResultsEffect of different salt diet on RAS of the OVLT in rats with chronic renal failure1. General data1.1Blood biochemical results before Stimulation10weeks after modeling, The rats were weighed and the blood pressure was indirectly measured by the tail-cuff method. Then we collected the blood of rats for serum creatinine and blood urea nitrogen measurement. We found that serum creatinine, blood urea nitrogen, arterial blood pressure and24-hour urine protein of the CRF rats were significantly higher and no body weight change compared with the sham group (Independent samples T-test, BW, t=1.559, P=0.14; Scr, t=-4.021, P=0.01; SBP, t=-2.597, P=0.02;24hU Protein, t=-6.686, P=0.01).1.2Blood biochemical results after Stimulation14days after Stimulation, In the Sham groups, compared with the normal salt,24-hour urinary sodium was significantly elevated in high-salt group and decreased in the low-salt group, However, the remaining did not change (One-Way ANOVA, Body weight, F=0.915,P=0.411; SBP, F=0.518, P=0.607; Serum Na+, F=1.926, P=0.175;24hU NA+, F=136.072, P=0.000;24hU Protein, F=1.477, P=0.26). In the CRF groups, compared with the normal salt, All parameters detected were significantly increased in high-salt group except for body weight and24hour urinary sodium; and24h urinary sodium and arterial blood pressure were significantly decreased in low-salt group(One-Way ANOVA, Body weight, F=0.056, P=0.946; SBP, F=18.250, P=0.000; Serum Na+, F=3.211, P=0.063;24hU Na+, F=113.316, P=0.000;24hU Protein, F=53.409, P=0.000).In the low salt intake groups, compared with the sham rats,24-hour urinary sodium excretion was significantly decreased in CRF rats, the remaining did not change (P<0.05). In the normal salt intake groups, compared with the sham rats,24-hour urinary protein excretion was significantly increased in CRF rats, the remaining did not change (P<0.05). In the high salt intake groups, compared with the sham rats, SBP, serum sodium,24-hour urinary sodium excretion was significantly increased and24-hour urinary sodium excretion was significantly decreased in CRF rats, the body weight did not change (P<0.05)2. Effect of different salt diet on renin-angiotensin system of OVLT in CRF rats2.1Effect of different salt diet on AT1receptor protein and mRNA expression of OVLT in CRF ratsIn the sham group, compared with the normal salt group, the protein expression of AT1receptor was increased in high salt group, but there was no statistically significant; and significantly decreased in low-salt group (P<0.05in Western blotting and Immunohistochemistry).In the CRF group, compared with the normal salt group, the protein expression of AT1receptor was significantly increased as increase of salt concentration (P<0.05in Western blotting and Immunohistochemistry). More importantly, compared with the corresponding salt of the sham group, AT1receptor expression of the CRF rats was significantly higher (P<0.05). In the sham group, compared with the normal salt group, the mRNA expression of AT1receptor was increased in high salt group, but there was no statistically significant, and significantly decreased in low-salt group,(ATI receptor Realtime PCR, One-Way ANOVA, F=10.535, P=0.011). In the CRF group, compared with the normal salt group, the mRNA expression of AT1receptor was significantly increased in high salt group, and decreased in low salt group, but there was no statistically significant (AT1receptor Realtime PCR, One-Way ANOVA, F=64.780, P=0.002) More importantly, compared with the corresponding salt of the sham group, AT1receptor expression of the CRF rats was significantly higher (P<0.05).2.2Effect of different salt diet on ANGⅡ protein expression of OVLT in CRF ratsIn the sham group, compared with the normal salt group, the protein expression of ANGⅡ was increased in high salt group, but there was no statistically significant; and significantly decreased in low-salt group (ANGⅡ Immunohistochemistry, One-Way ANOVA, F=20.423, P=0.002).In the CRF group, compared with the normal salt group, the protein expression of ANGⅡ was significantly increased as increase of salt concentration (ANGⅡ Immunohistochemistry, One-Way ANOVA, F=225.921,P=0.000). More importantly, compared with the corresponding salt of the sham group, ANGⅡ expression of the CRF rats was significantly higher (P<0.05).2.3Effect of different salt diet on ACE protein and mRNA expression of OVLT in CRF ratsIn the sham group, compared with the normal salt group, the protein expression of ACE was increased in high salt group, but there was no statistically significant, and significantly decreased in low-salt group (ACE Western blotting, One-Way ANOVA, F=6.434, P=0.001); In the CRF group, compared with the normal salt group, the protein expression of ACE was significantly increased as increase of salt concentration (ACE Western blotting, One-Way ANOVA, F=42.884, P=0.000). More importantly, compared with the corresponding salt of the sham group, ACE expression of the CRF rats was significantly higher (P<0.05). In the sham group, compared with the normal salt group, the mRNA expression of ACE was significantly decreased in low-salt group and increased in high salt group, but there was no statistically significant (ACE Realtime PCR, One-Way ANO VA, F=6.434, P=0.033);In the CRF group, compared with the normal salt group, the mRNA expression of ACE was significantly increased as increase of salt concentration (ACE Realtime PCR, One-Way ANOVA, F=28.603, P=0.001). More importantly, compared with the correspondin

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