Relationship Between Trimethylamine-N-Oxide And Aquaporin-2 In The Mechanism Of Hypertension

Cardiovascular disease is the leading cause of death in human beings currently,and hypertension is the most important independent risk factor for cardiovascular disease.Trimethylamine-N-oxide?TMAO?is a recently discovered marker of cardiovascular disease.The researchers found a correlation between elevated fasting plasma TMAO and increased risk of major adverse cardiovascular events.In recent years,some researches have initially explored the relationship between TMAO and the development of hypertension.The researchers found that normal rats input with AngII could significantly increase systolic blood pressure and diastolic blood pressure,but only appeared in the first 5 days of perfusion;however,the pressure-raising effect of the rats that those were co-infused with AngII and TMAO persisted until the end of the experiment?the experiment lasted two weeks?.This experiment showed that TMAO could synergize the boosting effect of Ang II,but the exact mechanism remains unclear.TMAO is a small organic compound whose chemical formula is?CH3?₃NO,which usually exists as a dihydrated,colorless solid material.Recent studies have found that intestinal flora can use substances that rich in choline or trimethylamine?TMA?structure,such as phosphatidylcholine,L-carnitine,etc.,to produce TMA,and then TMA is quickly oxidized by flavin containing mono-oxygenase?FMO?to generate TMAO in human body.The concentration of TMAO in blood increases after people eat foods such as meat,eggs,milk,and fish riching in L-carnitine and phosphatidylcholine.In the past,we thought that TMAO-rich seafood was an important source of protein and vitamins in the Mediterranean diet and was beneficial to the circulatory system.It has long been considered that TMAO was a waste of choline metabolism and was mainly excreted in the urine;however,many studies in recent years have shown that TMAO involved in many important biological functions from bacteria to mammals and other organisms.Recent clinical studies have shown that elevated levels of plasma TMAO are positively associated with increased risk of major adverse cardiovascular and cerebrovascular events such as myocardial infarction or stroke.On the other hand,TMAO is a penetrating agent that plays a protective role in the cell homeostasis of many animals and plants.We know that the ability to control the volume of cells is the key to the survival of unicellular and multicellular organisms.Osmotic agents are small molecules that cells use to maintain cell volume,and they play a key role in the cell's ability to adapt to osmotic pressure and hydrostatic pressure.Several types of penetrants that cells use to regulate their volume are including amino acids and their derivatives,polyols and sugars,methylamine and urea,etc.On the contrast to salt ions,most penetrants do not interfere with protein structure.In addition,some penetrants such as TMAO appear to stabilize protein structure and function by neutralizing protein perturbations which are caused by changes in osmotic pressure,hydrostatic pressure,or urea.It has been suggested that TMAO is used in deep-sea fish to counteract the destabilizing effect of osmotic pressure and hydrostatic pressure on proteins.We humans constantly change the environment to adapt to our own needs,but our body's tissues and organs are also constantly exposed to osmotic pressure and hydrostatic pressure because of the frequent changings in our blood pressure,the hydrostatic pressure in our body changes with blood pressure at every moment.This affects the structure and function of the heart,brain,kidneys,and cells of the arteries and veins.For example,in the cellular environment of human kidney medulla,in order to produce concentrated urine,the kidney accumulates sodium and urea in the medullary region to establish a high osmotic gradient,and its osmotic pressure is three to five times higher than the osmotic pressure of plasma.It has been reported that the medulla of the kidney also accumulates TMAO,which acts as a penetrating agent to protect renal cell proteins from interference caused by urea.The main function of the kidney is to regulate the balance of water and osmotic pressure in the body.Early studies found that water passes through the lipid bilayer structure of cell membranes in a simple diffusion manner,but the permeability of the descending branches and collecting ducts of the renal medullary can be as much as tens or even hundreds of times higher than other parts of the body.The rapid water transport function is not only inhibited by mercuric chloride,but also restored by?-mercaptoethanol,thus inferring the existence of a specific transbiological membrane water channel.The first selective aquaporin?AQP?was discovered in the mid to late1980s and was named aquaporin1?AQP1?.Subsequently,new members of the AQP family were discovered one after another.There are at least 13 AQPs?AQP0-AQP12?in mammals and the kidney is the tissue with the highest AQP content in the body.It expresses at least seven subtypes:AQP1,AQP-2,AQP3,AQP4,AQP6,AQP7,and AQP11..Among them,aquaporin-2?AQP-2?is mainly distributed in the renal collecting ducts,and AQP-2 is expressed at the terminal end of urine formation,and passes through vasopressin(Arginine Vasopressin,AVP;The diuretic hormone Antidiuretic Hormones?ADH?regulates AQP-2,a key aquaporin of the kidney.Previous studies have shown that AQP-2 is mainly distributed on the luminal cell membrane and vesicle membrane of the main cells of the renal collecting ducts,also known as collecting duct aquaporins?AQP-CD?,which plays an important role in the urine enrichment mechanism of the kidneys.effect.In the basal state,AQP-2 is mainly located in intracellular vesicles.Upon stimulation with AVP,AQP-2 is transferred from the intracellular portion of the cell to the apical end of the cytoplasmic membrane,which is achieved by the extracellular fusion of AQP-2 vesicles.After AVP stimulation,the water permeability of the cell membrane was regulated by the transport of AQP-2 to the apical membrane;once AQP-2 appeared in the apical membrane of the cell,water could easily be transported into the cell by the main cell,thereby achieving water reabsorption.Most previous studies focused on AQP-2 translocation to the apical membrane after AVP stimulation,which modulates the permeability of cell membranes to water;however,there has been a question here:if AQP-2 is only expressed at the top of the main cell Membrane,then a large amount of water enters the cell through the AQP-2 fast water channel expressed in the apical membrane,but if there is no good water to drain and absorb into the channel of blood,the main cells of the collecting tube can tolerate huge water entry in a short time The pressure will cause the cells to swell,damage the various organelles in the cells,and even lead to the death of the cells.Many studies focused only on the opening of the apical membrane AQP-2 in the collecting duct main cells,but did not pay attention to where the water entering the main cells of the collecting duct entered the blood.Subsequent studies have found that,in most cases,intracellular AQP-2 is transferred to the apical membrane of the host cell after hormone stimulation;and in the main cells of the renal collecting duct and connecting tube,it can be seen that AQP-2 is also located in the Basal cell membrane.A detailed immunohistochemical method including counting of gold particles under an immunoelectron microscope revealed that AQP-2 is localized along the basolateral membrane in Bracketboro rat collecting ducts and connective cells in both normal and absent AVP conditions.Axial heterogeneity was also observed.Further studies revealed that AQP-2 was mostly localized in the collecting ducts of the inner pith and the basolateral membrane of the connective tube cells.A few of them were located in the outer main cells of the cortex and medullary collecting ducts.Basal membrane.In this way,we can explain that AQP-2 is expressed not only in the apical membrane of the host cell,but also in the basolateral membrane of the main cell,which is equivalent to the establishment of a fast water channel that communicates with the vessel and the blood vessels in the collecting tube main cell.The AQP-2 enters the main cells of the collection tube and is then rapidly absorbed into the blood.This ensures the stability and balance of the volume,shape and function of the main cells of the collecting tube,and the rapid weight of water in the inner marrow collecting tube and connecting tube of the kidney absorbed.Studies have shown that there is aggregation of TMAO in the kidney,and whether the elevation of TMAO regulates the expression and opening of AQP-2,thereby regulating water metabolism and participating in osmotic pressure regulation and other functions?The relevant mechanisms of the two still need further study.In summary,TMAO is a small molecule of osmotic,aggregated in the kidney,and may be related to changes in osmotic pressure,excreted by the kidney;while TMAO has the role of stabilizing protein structure and function,recent studies suggest that TMAO can assist AngII plays a role in prolonging the duration of boosting;and AQP-2 plays a very important role in regulating water metabolism and maintaining the change of osmotic pressure.These important features indicate that the mechanism of blood pressure upregulation caused by TMAO may be closely related to the mechanism of AQP-2regulating water metabolism and maintaining the osmotic pressure.The purpose of this study was to investigate the plasma TMAO levels in spontaneously hypertensive rats and to evaluate the effect of TMAO on arterial blood pressure?BP?in spontaneously hypertensive rats;and to further explore the interaction between TMAO and AQP-2 in the development of hypertension at the cellular level.The study of the effects and molecular mechanisms may provide basic intervention strategies for the prevention and treatment of hypertension in clinical practice,advance the prevention of cardiovascular diseases,and reduce the incidence of hypertension and cardiovascular and cerebrovascular diseases.Part?Effect of Trimethylamine-N-oxide?TMAO?on the Expression of Aquaporin-2 in Kidney of Spontaneously Hypertensive RatsObjective:The purpose of this study was to investigate the plasma TMAO levels in spontaneously hypertensive rats?SHR?and the relationship between TMAO and AQP-2in the mechanism of hypertension.Methods:Twelve-week-old male spontaneously hypertensive rats?SHR,n=40?and Wistar-Kyoto rats?WKY,n=40?were divided into SHR and WKY groups.SHR rats were randomly assigned to four groups?10 in each group??1?SHR Untreated group:equal volume of normal saline was intragastrically administered as a control and administered once a day for oral administration;?2?SHR TMAO group:0.24%TMAO?ordinary feed containing 0.24%TMAO,wt/wt?was administered by oral gavage once a day;?3?SHR TMAO+TVP group:0.24%TMAO once daily by oral gavage;Vultan?Tolvapan,TVP,a V2 receptor antagonist?was intragastrically administered at 15 mg/kg twice daily;?4?SHR TVP group:TVP was intragastrically administered at 15 mg/kg,two daily This time,WKY rats were randomly divided into four groups?10 in each group?:?5?WKY Untreated group:equal volume of normal saline was intragastrically administered as a control and administered once a day orally;?6?WKY TMAO group:0.24%TMAO once daily by oral gavage;?7?WKY TMAO+TVP group:0.24%TMAO once daily by oral gavage;and TVP plus 15mg/kg gavage Drugs,twice daily;?8?WKY TVP group:TVP was administered intragastrically twice daily at 15 mg/kg.The test lasted for 6weeks.Blood pressure?BP?was measured every week.Blood samples of tail veins were collected weekly in fasted rats of SHR and WKY.Plasma TMAO expression was measured by liquid chromatography and triple quadrupole mass spectrometry.Plasma was detected.Osmotic pressure?POsm?,plasma vasopressin?PAVP?and plasma aquaporin-2?PAQP-2?concentrations were measured by ELISA,and the expression of AQP-2 in the kidney was detected by RT-PCR and Western blot.Results:From week 12 to week 17,there was no significant difference in plasma TMAO concentration between SHR Untreated and WKY Untreated rats.From week 14 to week17 of the experiment,plasma TMAO concentration was higher in SHR TMAO group compared with SHR Untreated group.,POsm,PAVP,and SBP all increased significantly;after intervention with TVP,compared with SHR TMAO group,plasma TMAO concentration,POsm,and SBP were significantly decreased in SHR TMAO+TVP rats,while PAVP appeared significantly.Increased,P<0.05,the difference was statistically significant.After TMAO intervention in SHR rats,the expression levels of PAQP-2,AQP-2 mRNA,and AOP-2 protein were significantly increased.After intervention of V2receptor antagonist TVP,PAQP-2 levels and AQP-2 mRNA were significantly increased.The expression levels of AOP-2 protein and AOP-2 protein were significantly decreased,P<0.05,and the difference was statistically significant.Similar trends were observed in the WKY group,but plasma TMAO concentrations,POsm,PAVP,and PAQP-2 in the WKY group were relatively late,but the trend was the same;RT-PCR and Western blotting in the WKY group.The expression of AQP-2 mRNA and AQP-2 protein in the medulla of kidney was detected by blot,but the expression of AQP-2 was less than that of SHR.Conclusion:An increase in TMAO levels in plasma leads to an increase in plasma osmolarity that triggers the release of AVP,leading to increased expression of AQP-2,resulting in increased reabsorption of water,leading to an increase in blood volume and eventually to an increase in blood pressure.Part ? The Mechanism of Action of Trimethylamine-N-Oxidation on Aquaporin-2Objective:HEK293 cells stably expressing AQP2 were established to further investigate the effect of TMAO on AQP2 expression in HEK293/AQP2,and the interaction mechanism between TMAO and AQP2.Methods:1.Stably expressed pc DNA3-AQP-2 cell line construction: The pc DNA3-AQP-2 plasmid was transfected into HEK293 cells using Lipofectamine 2000,and stable expression of pc DNA3-AQP-was confirmed by Western blot,RT-PCR and immunofluorescence.2 Cell lines were successfully constructed.2.Different concentrations of TMAO intervention stable expression of pc DNA3-AQP-2 cell line,grouped:?1?HEK293control group: intervention with concentration gradient?0,2,4,20,40,200,400??mol/L TMAO?2?HEK293/Veckor group: intervention with TMAO with a concentration gradient of?0,2,4,20,40,200,400??mol/L;?3?HEK293/AQP-2 group: with concentration gradient?0,2,4,20,40,200,400??mol/L of TMAO was intervened and HEK293 cells stably transfected with pc DNA3-AQP-2 were treated with different concentrations of TMAO to identify the most pro-AQP-2 expression.The concentration of TAMO,Western blot and RT-PCR were used to detect the expression of AQP-2.3.Under the influence of optimal TMAO concentration,V2 receptor antagonist TVP was given to further explore the interaction mechanism between TMAO and AQP-2.Grouping: Stably transfected HEK293/AQP-2 cells: Grouping:?1?control group: blank control Group,?2?TMAO group: 20 ?mol/L TMAO,?3?TVP + TMAO group: 10 ?M TVP + 20 ?mol/L TMAO,?4?TVP group: 10 ?M TVP,RT-PCR and Western blot techniques-The m RNA and protein levels of AQP-2,V2 receptor,c AMP,PKA,etc.were detected on HEK293/AQP-2 cells.Results:Western blot,RT-PCR and immunofluorescence showed that the pc DNA3-AQP-2 receptor was successfully expressed in HEK293 cell line,and HEK293 cell line stably expressing pc DNA3-AQP-2 was successfully established;stable expression of pc DNA3-AQP-2 was achieved.HEK293 cells were treated with different concentration of TMAO,and the expression of AQP-2 was increased with the increase of TMAO concentration;the difference was statistically significant?P<0.05?.However,when the TMAO concentration was as high as 200 ?mol/L,the transfected pc DNA3-AQP-2 and HEK293/Vector cell strains died.After selecting the optimal concentration of TMAO in the HEK293 cell line stably expressing pc DNA3-AQP-2,the expression of AQP-2,V2 receptor,c AMP,PKA and other m RNA and protein were increased in the TMAO group and pretreated by TVP.After minutes,TMAO was added,and the m RNA and protein expressions of AQP-2,V2 receptor,c AMP,PKA were decreased,and the difference was statistically significant?P<0.05?.Conclusion:The HEK293 cell line stably expressing pc DNA3-AQP-2 was successfully established.As the concentration of TMAO increased,the expression of AQP-2 was increased.However,when the concentration of TMAO was too high,the cell strain died and the concentration of TMAO increased,activating V2 of AQP-2.Receptors,which in turn promote the activation of intracellular c AMP-PKA pathway,increase the movement of vesicles containing AQP-2 to the surface of cell membranes,increase the expression of AQP-2 cell membrane,and increase the permeability of cells to water;when the concentration of TMAO is too high,water becomes transparent.Excessive increase in sex,leading to increased intracellular water loss,cell shrinkage,death;TVP can inhibit TMAO increased AQP-2 expression,and cell shrinkage and other phenomena have significantly reduced.