The Role Of Logistic Protein GS28 And Adiponectin Receptor 1 In Regulating Blood Pressure By Modulating Urinary Sodium Excretion

BackgroundHypertension is one of the most common diseases in the world and the leading cause of premature death among adults.7.6 million people in the world die of high blood pressure and94 million people are disabled by high blood pressure every year.According to the latest survey data of hypertension in China,the crude prevalence of adult hypertension in China from 2012 to 2015 is 27.9%,and 71%of stroke and 54%of myocardial infarction death in China are related to hypertension.Evidence-based medicine shows that lowering blood pressure can significantly reduce the occurrence of cardiovascular events.Although people have made great achievements in the study of hypertension,the etiology of hypertension is still unknown,and it is of great significance to make clear the mechanism of hypertension.The pathogenesis of hypertension is complicated and involves many tissues and organs including central nervous system,heart,kidney and blood vessel.Although its mechanism is unknown,the function of urinary sodium excretion in kidney has been paid great attention.Kidney is the main organ of regulating the metabolism of water and sodium,and urinary sodium excretion is closely related to the blood pressure level of the body.Under physiological conditions,the amount and intake of sodium salt are almost equal,which is called sodium balance,which is an important mechanism for the body to maintain the stability of the internal environment and ensure the stability of blood pressure.Increased renal sodium reabsorption leads to decreased urinary sodium excretion,which in turn leads to sodium retention and elevated blood pressure.The proximal convoluted tubules of the kidney are the important parts responsible for the reabsorption of urine sodium,accounting for 65-70%of the reabsorption of urine sodium in the kidney.In recent years,as an important substance regulating the transport of substances,the role of intracellular logistics proteins has been paid attention to.such as:in renal collecting duct epithelial cells,logistics molecules SNAREs?SNAP-23 assist in targeting H-ATP enzymes to the apical membrane to complete H transmembrane transport;vesicle transport is also the way parathyroid hormone induces enzyme endocytosis in hamster kidney cells.The proximal tubule(renal proximal tubule,RPT)of the kidney is the main part of sodium resorption.In order to explore the function and mechanism of logistics system,we compared and analyzed the differential expression of renal tissue logistics molecular RNA between WKY rats and SHR rats by using a RNA chip.Obesity and hypertension are closely related.About 78%of men with hypertension and65%of women with hypertension have a cause directly related to obesity.Individual systolic and diastolic blood pressure are positively correlated with body mass index or waist circumference.Most obese hypertension patients with weight loss can have a decrease in blood pressure.Fat cells are widely distributed in the human body,not only an energy storage organ,but also an organ with endocrine function,which maintains a close relationship with the central nervous system,immune system and other endocrine organs by secreting a variety of fat factors.The role of adiponectin has been concerned.Our previous study found that adiponectin receptor is abundant in RPT cells,meanwhile,renal artery perfusion of adiponectin can significantly promote urinary sodium excretion in WKY rats,but this diuretic natriuretic effect is obviously impaired in SHR rats.further studies found that decreased adiponectin sensitivity in hypertensive rats was due to excessive phosphorylation of Adipo R₁in SHR rats.But Adipo R1 specific phosphorylation sites are unclear,and what causes Adipo R₁ hyperphosphorylation is unclear.Aims:1.To study whether GS28 kidney is abnormally expressed in hypertensive state.2.To study whether GS28 abnormal expression in the kidney is associated with elevated blood pressure.3.The mechanisms of GS28 effects on blood pressure.4.To study adipo R₁ phosphorylation sites.5.Further clarify the key role of GRK4 in Adipo R₁ phosphorylation.Methods:1.To study whether GS28 kidney is abnormally expressed in hypertensive state.(1)Selecting differentially expressed logistics molecules between SHR and WKY through the results of kidney gene chip,and then determining the differentially expressed logistics molecules by RT-PCR.(2)Confirming the up-regulation of GS28 expression in SHR by RT-PCR and WB.(3)To confirm the higher expression of GS28 in human hypertension-RPT cell lines.2.To study whether GS28 abnormal expression in the kidney is associated with elevated blood pressure.(1)Knock down GS28 by UTMD,monitor the blood pressure by tail-cuff method,and the urine volume and urine sodium were monitored by metabolic cage.(2)Knock down GS28 in RPTcells by si-GS28,measure Na⁺-K⁺-ATPase activity.3.The mechanisms of GS28 effects on blood pressure.(1)To select the proteins linked to GS28 by protein mass spectrometry,immunoprecipitation and laser confocal.(2)Knock down GS28 by si RNA,and measure the Na⁺-K⁺-ATPase activity,and to study the changes of distribution of NAK?1 by immunoblotting,immunofluorescence and immunohistochemistry.(3)To study the phosphorylation of NKA?1 after si GS28 interference by Western blotting.4.To study adipo R₁ phosphorylation sites.(1)To predict the phosphorylation site of Adipo R₁ by bioinformatics and to prepare mutant physique grains.(2)To study the efficiency of plasmid transfection by RT-PCR and Western blot.(3)To study the diffrences of Adipo R₁ phosphorylation and the binding of Adipo R₁ and G?i between wildtype and mutant plasmid transfected RPTcells by immunoprecipitation.(4)To study the different responses to adiponectin between the wild-type or mutant plasmid transfected RPT cells by measuring the Na⁺-K⁺-ATPase activity.5.Further clarify the key role of GRK4 in Adipo R₁ phosphorylation.(1)Knock down the GRK4 by UTMD,and to study the transfecting efficiency by RT-PCR and Western blot.(2)To study the phosphorylation of Adipo R₁ and the binding of Adipo R₁ and G?i by immunoprecipitation.(3)To study the diuretic and natriuretic effects of adiponectin afer GRK4 knock down by single renal perfusion.Results:1.The GS28 in the renal cortex of SHR rats was significantly higher than that of WKY rats at the same age.2.The GS28 protein level in hypertensive human RPT cells was significantly higher than that in normal human RPT cells.3.Increased natriuresis and decreased blood pressure were observed in SHR whose GS28 of renal were knocked down,suggesting that GS28 plays an important role in the development of hypertension.4.The Na⁺-K⁺-ATPase activity of RPTcells was decreased after GS28 were knocked down,suggesting that GS28 affects the resorption of water and sodium by affecting Na⁺-K⁺-ATPase activity.5.GS28 have direct connection with NKA?1,suggesting that GS28 could modulate Na⁺-K⁺-ATPase activity directly.6.GS28 could affect the intracellular distribution of the NKA?1.7.GS28 modulate NKA?1 distribution through modulating its phosphorylation.8.The decrease of the phosphorylation levels of Adipo R₁ and the increase of the bingding of Adipo R₁ and G?i were observed after Ser7?Thr24?Thr43 were mutated to Ala.9.Sensitive to adiponectin was restored after SHR RPT cells were transfected by mutant plasmid.10.The decreacse of blood pressure and increase of urinary sodium excretion were observed after GRK4 in kidney was knocked down in SHR..11.The decrease of the phosphorylation levels of Adipo R₁ and the increase of the bingding of Adipo R₁ and G?i were observed after renal-GRK4 was knocked down in SHR.12.Sensitive to adiponectin was restored after renal-GRK4 was knocked down in SHR.Conclusion:1.High expression of GS28 in SHR RPT cells cause hypertension by increasing Na⁺-K⁺-ATPase activity.2.GS28 regulates the intracellular distribution by affecting the phosphorylation of NKA?1,and then affects the Na⁺-K⁺-ATPase activity.3.The excessive phosphorylation of Adipo R₁ in SHR RPT cells leads to desensitization to adiponectin,Ser7?Thr24?Thr43 were the key phosphorylation sites.4.GRK4 leads to excessive phosphorylation of Adipo R_1,and knocking down GRK4could restore the sensitivity to adiponectin.