Salt-induced Phosphoproteomic Changes In The Hypothalamic Paraventricular Nucleus And Subfornical Organ In Rats With Chronic Renal Failure

BackgroundChronic kidney disease(CKD)has become increasingly considered as one of a global public health problem.When chronic kidney disease express as deterioration of renal function,disease progressing to the occurrence of metabolic disorders and clinical symptoms of the syndrome,it will be defined as chronic renal failure(CRF).As early as 1998,Coresh J and others pointed out that chronic renal insufficiency in patients with high blood pressure is a frequent cardiovascular event.In individuals who with chronic kidney disease,the risk and mortality of cardiovascular disease is significantly increased.In traditional cardiovascular risk factors,impaired renal function and increased urinary albumin can increase the risk of developing cardiovascular disease 2-4 times,forming a vicious circle.Therefore,in the treatment of chronic renal failure,active control of blood pressure is an important treatment measures.The central nervous system(CNS)has played an important role in the formation and development of renal hypertension and the pathophysiological changes of chronic kidney disease.Central nucleus such as subfornical organ(SFO),hypothalamic paraventricular nucleus(PVN),the rostral ventrolateral medullary nucleus(RVLM),and the nucleus of solitary tract(NTS),which are responsible for the regulation of neurogenic hypertension and renal afferent activity.There are local RAS in the brain SFO and PVN,the activation of its RAS can activate the sympathetic nervous system so as to increase blood pressure and regulate the function of peripheral organs such as kidney.Similarly,in the condition of chronic renal failure,it can also activate the SFO and PVN neurons,inducing inflammatory infiltration and sympathetic activation.Not only that,a large number of uremic toxins produced in CKD can also promote neurohumoral dysfunction by modulating brain cell and molecular pathways and increasing the sensitivity of the central nervous system to extracellular signals.Nearly 50%of patients who with essential hypertension have salt sensitivity,and CKD patients also have salt sensitivity.Increased intake of habitual dietary salt leads to increased systemic blood pressure and glomerular pressure which can enhance the risk of proteinuria,night blood pressure drops which accompanied by impaired renal function,and eventually developed into end-stage renal disease(ESRD).On the other hand,high salt diet can also increase cerebrospinal fluid Na+concentration and the sympathetic nervous system activity in salt-sensitive rat,and finally leads to the high blood pressure.The progress of CKD caused by high salt intake can cause the dysfunction of PVN and SFO,manifested as inflammatory infiltration,increased reactive oxygen species,local renin-angiotensin system activation and excessive secretion of arginine vasopressin.Proteomics essentially refers to the ability to study protein at a large scale,based on protein biodiversity,including protein expression levels,protein subtypes,protein translational modifications,and protein-protein interaction,etc.,so that it can obtain the overall and comprehensive understanding of the occurrence of disease and cell metabolism and other pathophysiological processes.Protein phosphorylation is a post-translational modification that plays an important role in cell regulation and signal transduction,which involves the entire cellular process from cell proliferation,differentiation to apoptosis.Protein phosphorylation plays a key role in the function regulation of PVN and SFO,it involves energy metabolism,activation of RAS and sympathetic activation,synaptic transmission and secretion of neurotransmitters.Therefore,we should not only focus on the analysis of protein expression level in different pathophysiological conditions,protein phosphorylation modified signal transduction is also associated with the further development of the disease.The aim of the present study was to profile large-scale phosphoproteomic analysis of PVN and SFO induced by CRF and high salt-load by using relative absolute quantification(iTRAQ)techniques combined with Liquid chromatography-tandem mass spectrometry(LC-MS/MS)and TiO2 enrichment.Chapter 2 Salt-induced phosphoproteomic changes in the hypothalamic paraventricular nucleus in rats with chronic renal failureMethod1.Construction of animal model and collection of tissue samples(1)Experimental animal purchase and breeding;(2)5/6 nephrectomy(5/6Nx)established chronic renal failure model;(3)Orbital vein blood collection and testing the blood pressure by tail;(4)Rats were randomly divided into 3 groups and stimulated with different concentrations of salt diet for 3 weeks;(5)Rat blood and fresh brain tissue collection and treatment;(6)Serum creatinine test.2.The phosphorylated proteomics of PVN was analyzed by iTRAQ(isobaric tags for relative and absolute quantification)technique(1)Protein extraction;(2)SDS-PAGE electrophoresis;(3)Enzyme digestion and peptide quantification;(4)Peptide labeling;(5)Titanium dioxide enrichment;(6)Mass spectrometry analysis;(7)Data analysis;(8)Verified the altered phosphorylated protein using Western Blotting.Results1.Establishment of the Chronic Renal Failure Rat ModelBlood creatinine and blood pressure were significantly higher in the 5/6Nx rats than in the sham operation group(p<0.05).2.Changes of blood pressure and serum creatinine after administration of different salt dietThe blood pressure and serum creatinine of 5/6Nx rats were significantly higher than the sham group(p<0.05).In the 5/6Nx rats,the blood pressure of the high salt diet group was significantly higher than that of the normal salt diet group(p<0.05).3.Changes of phosphorylated peptides in PVN after administration of different concentrations of salt diet(1)Quantitative results of phosphorylated peptidesIn this study,3723 phosphorylated peptides corresponding to 1530 phosphorylated proteins were identified by iTRAQ technology,among these 3719 phosphorylated peptides were quantified.In normal salt diet,5/6Nx rats induced 274 phosphorylated peptides(133 up-regulated and 141 down-regulated)which differentially expressed compared with sham rats(NC/NS).In chronic renal failure rats,high salt diet resulted in a change of 302(160 up-regulated,142 down-regulated)phosphorylated peptides compared to normal salt diet(HC/NC).(2)The peptide phosphorylation site distributionA total of 4548 phosphorylation sites with high confidence were identified.Among them,the number of serine,threonine,tyrosine were 4019,513,16 respectively.(3)Gene Ontology(GO)Classification of Total Phosphorylated ProteinsThe major biological processes of phosphorylated proteins in PVN include cell processe,single-organism processe,biological regulation,and so on.(4)Gene Ontology(GO)Classification of Differential Phosphorylated ProteinsIt indicated that whether the NC/NS groups or HC/NC groups,the main biological process which the differential phosphorylated protein involved in was including the cell process,single-organism process and biological regulation.Its molecular function was mainly including binding,transport activity,catalytic activity and so on.(5)STRING protein-protein analysis of differentially phosphorylated proteinsIn the NC/NS comparison groups,among the 211 phosphorylated proteins,25 differential proteins could functionally linked directly or indirectly.The most linked phosphoproteins were Map la,Hsp90aal,Dpysl3,Synl,Atplal,Atpla2 and Camk2a.There were 24 differential proteins could functionally linked directly or indirectly in the HC/NC comparison groups of the 224 phosphoproteins.The most linked phosphorylated proteins were Mapk3,Mapla,Camk2a,Synl,Dnml and Map2.(6)KEGG signaling pathway analysis of differential phosphorylated proteinThe results showed that there were cAMP signaling pathway,tight junction,endocrine,insulin secretion,Ras signaling pathway,Mapk signal pathway and PI3K-Akt signaling pathway etc.in NC/NS comparison groups.The function of these pathways involved in cell proliferation,cell communication,apoptosis,energy metabolism,neurotransmitter secretion,nerve signal conduction..In HC/NC comparison groups,the differential phosphorylated protein is mainly involved in cAMP signaling pathway,calcium signaling pathway,insulin secretion,Mapk signal pathway,PI3K-Akt signaling pathway,aldosterone synthesis and secretion.Its function involved in cell proliferation,cell communication,apoptosis,Energy metabolism,neurotransmitter secretion,nerve signal transmission and so on.(7)Protein expression of phospho-dynaminl and phospho-TPPP in PVN of rats with chronic renal failure and salt loadThe results of immunoblotting showed that the expression levels of phospho-dynaminl and phospho-TPPP of CRF rats in the high salt group were significantly higher than those in the normal salt group(p<0.05),which was consistent with the results of protein mass spectrometry.Conclusion1.In this study,we used iTRAQ technique to construct the phosphorylated proteomics of PVN induced by high salt and chronic renal failure.A total of 3723 phosphorylated peptides corrsponding to 1530 phosphorylated proteins were identified,among these 3719 phosphorylated peptides were quantitatied.2.Compared with the sham group,chronic renal failure resulted in 133 upregulated and 141 downregulated of the phosphorylated peptides under the normal salt diet.In chronic renal failure rats,compared with the normal salt diet,the high salt diet induced 160 upregulated and 142 downregulated of the phosphorylated peptides.3.The biological function of the identified phosphorylated proteins were mainly including binding,transport activity,catalytic activity and other important functions.Chapter 3 Salt-induced phosphoproteomic changes in the subfornical organ in rats with chronic renal failureThe research method is the same as above.Results1.To confirm the successful establishment of the rat model of chronic renal failureThe serum creatinine and blood pressure of 5/6Nx rats were significantly higher than that in sham operation group(p<0.05),but there was no significant difference in body weight.2.Changes of blood pressure and serum creatinine after administration of different salt dietHigh salt diet induced a significantly higher blood pressure and serum creatinine in 5/6Nx rats than that with normal salt diet(p<0.05).In the 5/6Nx rats,the blood pressure of the high salt diet group was significantly higher than that of the normal salt diet group(p<0.05).3.Changes of phosphorylated peptides in SFO after administration of different concentrations of salt diet(1)Quantitative results of phosphorylated peptidesIn this study,6808 phosphorylated peptides corresponding to 2661 phosphorylated proteins were identified by iTRAQ technology,among these 6719 phosphorylated peptides were quantified.In normal salt diet,5/6Nx rats induced 418 phosphorylated peptides(168 up-regulated and 250 down-regulated)which differentially expressed compared with sham rats(NC/NS).In chronic renal failure rats,high salt diet resulted in a change of 278(154 up-regulated,124 down-regulated)phosphorylated peptides compared to normal salt diet(HC/NC).(2)The peptide phosphorylation site distributionA total of 11992 phosphorylation sites with high confidence were identified by Proteome Discoverer 1.4 software.Among them,the number of serine,threonine,tyrosine were 10474,1417,101 respectively.(3)Gene Ontology(GO)Classification of Differential Phosphorylated ProteinsThe major biological process of the differential phosphorylated protein in NC/NS or HC/NC comparison groups were cell process,single-organism process,biological regulation,biological process regulation,stress response,and so on.Molecular function analysis of differential phosphorylated protein showed that the molecular function of NC/NS or HC/NC comparison groups were mainly including binding,catalytic activity,molecular function regulation,transport activity,structural molecular activity and so on.(4)STRING protein-protein analysis of differentially phosphorylated proteinsIn the NC/NS comparison groups,51 differentially phosphoproteins could functionally linked directly or indirectly.The most linked phosphoproteins were Dlgapl,Syp,Synl,Mapla,Stx1a.There were 22 differential proteins could functionally linked directly or indirectly in the HC/NC comparison groups.The most linked phosphorylated proteins were Dlg4,Dlg2,Map1a,Map2,Syngapl.(5)KEGG signaling pathway analysis of differential phosphorylated proteinsKEGG pathway analysis showed that the phosphorylated proteins was mainly involved in the signal pathway such as cAMP signal pathway,Mapk signal pathway,tumor pathway,Wnt signaling pathway.Its function involved in cell proliferation,cell communication,apoptosis,energy metabolism,neurotransmitter secretion,nerve signal transmission and so on.The expression of phosphor-?-catenin and phosphor-Hsp27 in the Wnt/?-catenin signaling pathway and the p38/Hsp27 signaling pathway were also found.Conclusion1.In this study,we used iTRAQ technique to construct the phosphorylated proteomics of SFO induced by high salt and chronic renal failure.A total of 6808 phosphorylated peptides corrsponding to 2661 phosphorylated proteins were identified,among these 6719 phosphorylated peptides were quantitatied.2.Compared with the sham group,chronic renal failure resulted in 168 upregulated and 250 downregulated of the phosphorylated peptides under the normal salt diet.In chronic renal failure rats,compared with the normal salt diet,the high salt diet induced 154 upregulated and 124 downregulated of the phosphorylated peptides.3.The biological function of the identified phosphorylated proteins were mainly including binding,catalytic activity,molecular function regulation,transport activity and some other important functions.