| Studies at home and abroad have shown that the hyperactivation of renal sympathetic nerves plays an important role in the occurrence and maintenance of essential hypertension.An important feature of the hyperactivation of sympathetic nerves is the increased content of catecholamines such as epinephrine and norepinephrine.Such sympathetic neurotransmitters act on the body’s α and βadrenergic receptors not only increase cardiac contractility,accelerate heart rates,but also increase the water and sodium reabsorption effects of kidney,enhance water and sodium retention effects of our body,and ultimately lead to hypertension.Among them,β2 adrenergic receptors(ADRB2)are widely distributed in almost all nephron segments,especially the proximal convoluted tubules.Sodium-hydrogen exchanger(NHE)is an important channel protein for water and sodium reabsorption by the kidneys.NHE1 and NHE3 are two major subtypes highly expressed in kidney tissue.Most of the crude urine that filters through glomerulus is reabsorbed through NHE3 in the apical membrane of proximal tubule cells.Activating ADRB2 enhance the activity of the NHE of renal tubular epithelial cells,increase the reabsorption effects of water and sodium through renal tubular epithelial cells,participate in the progress of water and sodium retention,and then affect the occurrence of hypertension,but its molecular mechanism is not currently clear.Some studies believe that this process does not rely on traditional G protein coupling signals.In this study,the effects of the β-adrenergic receptor agonist-isoproterenol(ISO)on the intracellular p H(p Hi)recovery rate and the expression of NHE1/NHE3 protein in the human proximal tubule cells line HK-2 were examined to explore the effect of ISO on the activity and protein expression of NHE in human renal tubular epithelial cells.The faster the recovery rate of p Hi,the stronger the Na+/H+ exchange activity of NHE.After a certain time of activation by agonist,the cytoplasmic tail of ADRB2 is phosphorylated by G protein coupled receptor kinase 2(GRK2)lead to desensitization of traditional G protein coupling signal;the phosphorylated cytoplasmic tail binds to β-arrestins(ARRBs)which mediated the transmission of non-G protein coupling signal,eventually causes a series of cellular response.This study assumes that ISO regulates the expression and activity of NHE through GRK2/ARRB1 mediated non-G protein-coupled signaling pathway;we explore the molecular mechanism by which ISO regulates the expression and activity of NHE in HK-2 cells through GRK2 inhibitor and propranolol drug intervention,and the method of constructing stably transfected cell line by lentivirus for ARRB1 overexpression.The molecular mechanism by which ISO regulates the expression and activity of NHE will provide a theoretical reference for improving the understanding of pathogenesis of hypertension and provide a potential target for the treatment of essential hypertension.Part 1 The effect of ISO on NHE activity and protein expressionObjective: To observe the effect of ISO on the NHE activity and the protein expression of NHE1/NHE3 and ADRB2 in human renal tubular epithelial cells.Methods: The human renal tubular epithelial cell line HK-2 was divided into ISO group and control group.They were treated with 50 μmol/L ISO and the same dose of saline for 24 hours respectively,and then the p Hi recovery rate and NHE1/NHE3,ADRB2 protein expression of each group were detected.Results:(1)The recovery rate of p Hi in the ISO group was significantly higher than that in the control group,and the differences was statistically significant(P < 0.05).(2)The expression levels of NHE1 and NHE3 proteins in the ISO group were higher than those in the control group,and the differences was statistically significant(P < 0.001).(3)The expression of ADRB2 protein in the ISO group was lower than that in the control group,and the differences was statistically significant(P < 0.01).Conclusions: The β2 adrenergic receptor agonist ISO can significantly enhance the NHE activity of human renal tubular epithelial cells,increase the NHE1/NHE3 protein expression,and reduced the ADRB2 protein expression level.Part 2 The effect of G protein-coupled receptor kinase 2(GRK2)inhibitor on ISO regulation of NHE activity and expressionObjective: To understand the effect of ISO on cell GKR2 enzyme activity,and to observe the effect of GRK2 inhibitor on HK-2 p Hi recovery rate,NHE1/NHE3 and ADRB2 protein expression.Methods:(1)HK-2 was divided into control group,ISO group,ISO+GRK2inhibitor group and GRK2 inhibitor group.They were given saline,50 μmol/L ISO,50 μmol/L ISO+300 nmol/L GRK2 inhibitor,and 300 nmol/L GRK2 inhibitor for 24 hours respectively,the GRK2 enzyme activity of each group of cells was detected.(2)The grouping was the same as above,and the recovery rate of p Hi,NHE1 and NHE3 protein expression and ADRB2 protein expression of each group are detected respectively.(3)HK-2 was divided into control group,ISO group,ISO+propranolol group,and propranolol group.They were given normal saline,50 μmol/L ISO,50 μmol/L ISO+50 μmol/L propranolol and 50 μmol/L propranolol for 24 h respectively,the recovery rate of p Hi of cells in each group was then detected.Results:(1)The differences of GRK2 activity among the control group,ISO group,and ISO+GRK2 inhibitor group was statistically significant(P < 0.001),among which the ISO group was the highest,the control group and ISO+GRK2inhibitor group followed,and the GRK2 inhibitor group was the lowest.(2)The differencess of p Hi recovery rate among control group,ISO group,ISO+GRK2inhibitor group and GRK2 inhibitor group was statistically significant(P < 0.001),among which the ISO group was the highest,ISO+GRK2 inhibitor group followed,control and GRK2 inhibitor group were the lowest.(3)The relative protein expression levels of NHE1/NHE3 in the control group,ISO group,ISO+GRK2 inhibitor group and GRK2 inhibitor group were significantly different(P < 0.001),among which the ISO group was the highest,ISO+GRK2inhibitor group followed,control and GRK2 group were the lowest.(4)The differences of ADRB2 protein expression among control group,ISO group,ISO+GRK2 inhibitor group and GRK2 inhibitor group was statistically significant(P <0.001),among which the ISO group was the lowest,ISO+GRK2inhibitor followed,and the control group and GRK2 inhibitor group were the highest.(5)The differences in p Hi recovery among the control group,ISO group,ISO+propranolol group and propranolol group was statistically significant(P <0.01),among which the ISO group was higher than the other groups.There was no statistical differences among the control group,the ISO+GRK2 inhibitor group and the GRK2 inhibitor group.Conclusions:(1)ISO increases the activity of GRK2.GRK2 inhibitors alleviate the activity of GRK2 after ISO treatment and also decreased the activity of GRK2 in control group.(2)ISO promotes the increase of NHE expression and activity,and reduceds the expression of ADRB2;GRK2 inhibitors can partially antagonize the regulatory effect of ISO on the functional activity of NHE,NHE and ADRB2 protein expression,but not on the NHE activity,NHE and ADRB2 protein expression of normal control cells.(3)Propranolol can completely block the positive regulatory effect of ISO on NHE activity.Part 3 The effect of overexpression of beta-Arrestin 1(ARRB1)on ISO-mediated regulation of NHE activity and expressionObjective: To understand the effect of ISO on ARRB1 protein expression and the role of GRK2 inhibitors in it,to observe the effect of overexpression of ARRB1 on NHE activity,NHE1/NHE3 and ADRB2 protein expression.Methods:(1)HK-2 was divided cells into control group,ISO group,ISO+GRK2 inhibitor group and GRK2 inhibitor group,and then WB and immunohistochemistry were used to detect the expression of ARRB1 and GRK2 proteins in each group.(2)HK-2 was divided cells into control group,ISO group,ISO+propranolol group and propranolol group,and then WB and immunohistochemistry were used to detect the expression of ARRB1 and GRK2 proteins in each group.(3)HK-2 was divided into control group,ISO group,ISO+ARRB1 overexpression group,ARRB1 negative overexpression group.control and ISO group were normal cells treated with normal saline and 50 μmol/L respectively,ISO+ARRB1 overexpression group and ARRB1 overexpression group were ARRB1 overexpression lentivirus infected cells treated with 50μmol/L ISO and normal saline respectively,ARRB1 negative overexpression group was control lentivirus infected cells treated with normal saline.After 24 h,the recovery rate of p Hi and the protein expression of NHE1/NHE3 and ADRB2 in each group were then tested respectively.Results:(1)The relative expression levels of ARRB1 protein in control group,ISO group,ISO+GRK2 inhibitor group and GRK2 inhibitor group are statistically significant(P <0.001),among which the ISO group was the lowest,followed by the ISO+GRK2 inhibitor group.The control group and the GRK2 inhibitor group were the highest.There was no statistical differences in GRK2 protein expression in each group.(2)The relative expression levels of ARRB1 protein in control group,ISO group,ISO+propranolol group and propranolol group are statistically significant(P <0.001),among which the ISO group was the lowest,the expression of ARRB1 among the other groups didn’t significantly differed from each other.(3)The differences of p Hi recovery rate among control group,ISO group,ISO+ARRB1 overexpression group,ARRB1 overexpression group and ARRB1 negative overexpression group was statistically significant(P <0.001),among which ISO group was the highest,control group and ARRB1 negative overexpression group followed,ISO+ARRB1 overexpression group and ARRB1 overexpression group were the lowest.(4)The relative expression levels of NHE1/NHE3 protein among control group,ISO group,ISO+ARRB1overexpression group,ARRB1 overexpression group and ARRB1 negative overexpression group were statistically significant(P <0.001),among which the ISO group was the highest.The control group and ARRB1 negative overexpression group followed,ISO+ARRB1 overexpression group and ARRB1 overexpression group were the lowest.(5)The relative expression levels of ADRB2 protein among control group,ISO group,ISO+ARRB1 overexpression group,ARRB1 overexpression group and ARRB1 negative overexpression group were statistically significant(P < 0.001),of which control group and ARRB1 negative overexpression group was the highest,followed by the ISO group,the ISO+ARRB1 overexpression group and the ARRB1 overexpression group were the lowest.Conclusions:(1)ISO significantly reduces the expression of ARRB1 in renal tubular epithelial cells.GRK2 inhibitors partially suppressed this effect,while propranolol mostly block the effects of ISO on ARRB1 expression.(2)ISO,GRK2 inhibitor and propranolol didn’t significantly change the protein expression of GRK2.(3)Overexpression of ARRB1 reduces NHE activity and protein expression,also decreased ADRB2 protein expression. |
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