The Role Of Adiponectin Receptor And Dopamine D₁-like Receptor In Regulating Blood Pressure By Promoting Urinary Sodium Excretion

BackgoundHypertension is a major modifiable risk factor for cardiovascular and renal diseases.About 7.6 million premature deaths each year and 92 million disability-adjusted life years are attributed to high blood pressure in the world.However,the mechanisms of hypertension are still not clear.The present study is of the view that the poor dialogue between the organs of the tissue is involved in the pathogenesis of hypertension.More and more studies have shown that fat is not only an energy storage tissue,but also an important endocrine organ in the body.Adipose tissue produces a variety of cytokines,including adiponectin?Adn?,leptin,resistin,interleukin-6 and so on,wherein the adiponectin and leptin have an effect in promoting the sodium excretion,and the resistin inhibits the excretion of sodium.Leptin did not decrease in the condition of high blood pressure,but increased;on the contrary,adiponectin decreased significantly in the patients with high blood pressure.In the patients with high blood pressure,the level of adiponectin in blood was negatively correlated with the increase of blood pressure.Adiponectin knockout mice(Adn^-/-)had high blood pressure induced by high-salt and high-fat diet,with a significant decrease in the expression of endothelial nitric oxide synthase in the vascular tiss ue;and hypertensive animal model,KKAy mice and SHR?Spontaneously hypertensive rats?rats treated with adiponectin can significantly reduce their blood pressure.The above results suggest that adiponectin plays an important role in the treatment of hypertension.The kidney plays an important role in regulating the balance of sodium and salt metabolism,and abnormal urinary sodium excretion is involved in the occurrence and development of hypertension.The hypertensive patients with significantly reduced adiponectin were associated with impaired urinary sodium excretion,and plasma adiponectin levels decreased in SHR?Spontaneously hypertensive rats?rats,accompanied by significant urinary sodium excretion disorders.Adiponectin,as a signaling molecule between adipose tissue and kidney,is not completely clear about the role of adiponectin in urinary sodium metabolism.We hypothesized that adiponectin promotes urinary sodium excretion under normal physiological conditions,and the absence of adiponectin-induced urinary sodium excretion is an important mechanism of hypertension.We constructed adiponectin knockout mice and wild-type control mice,and studied the phenotypes of adiponectin knockout mice,such as blood pressure,urinary sodium excretion.Dopamine receptors play an important role in promoting sodium excretion and regulating blood pressure,they are classified into two subtypes:D₁-like receptors,which include D₁R and D₅R,and D₂-like receptors,which include D₂R,D₃R,and D₄R.The role of D₁R is particularly prominent,the damage or loss of D₁R-mediated urinary sodium excretion function is an important factor in the occurrence of hypertension.In addition to phosphorylation desensitization,it was found that the absence of synergistic action between receptors is also an important way to affect the function of D₁R:CCK_BR?gastrin receptor?and D₁R can form heterodimers and there are physiological co-links which promote urinary sodium excretion.The absence of synergetic effect between CCK_BR and D₁R may be one of the important reasons for the impairment of D₁R function in SHR.In hypertensive animal model Zucker rats,previous studies have found that D₁R-mediated natriuretic excretion is also impaired,but the cause of D₁R dysfunction is not completely clear.This may be related to its reduced adiponectin content and its role in promoting urinary sodium excretion.Adiponectin,as a potential adipose factor that promotes the excretion of sodium in urine,may play its role through adiponectin receptors?AdipoRs?in the kidney.We speculate that AdipoR and D₁R can promote urinary sodium excretion in a synergistic manner.The decrease of synergistic effect between AdipoR and D₁R may be an important mechanism of D₁R dysfunction in Zucker rats,and is also one of the important phathogensis of essential hypertension.The D₅R is widely expressed in the mammalian kidney,specifically in the proximal tubule?PT?,thick ascending limb of Henle,distal convoluted tubule,and cortical collecting duct.Compared with the other dopamine receptors,D₅R has the highest affinity for dopamine and exhibits constitutive activity,which can be activated in the absence or presence of low concentrations of endogenous agonists.The D₅R,as with the other dopamine receptor subtypes,plays a vital role in the normal maintenance of body sodium and blood pressure by its own actions and interactions with other dopamine and G protein-coupled receptors?GPCRs?.Disruption of Drd5 in mice produces hypertension which is aggravated by a high salt diet.The renal expressions of the Ang II type 1 receptor?AT₁R?and renal sodium transporters are also increased in Drd5^-/-mice.In addition,Drd5^-/-mice have increased oxidative stress.The D₅R decreases reactive oxygen species?ROS?production by inhibition of phospholipase D?PLD?and nicotinamide adenine dinucleotide phosphate?NADPH?oxidase expression and activity,and upregulation of heme oxygenase-1?HO-1?.?/?hydroxylase 1 may also be involved in the D₅R-mediated regulation of ROS production.Humans have single nucleotide polymorphisms?SNPs?in the DRD5 gene,some of which have diminished D₅R function and abnormal coupling with adenylyl cyclase.The human D₅R F173L(hD₅R^F173L)mutation has a markedly impaired ability to stimulate cAMP production.To investigate the role of hD₅R^F173L in the development of hypertension,we generated hD₅R^F173L transgenic(hD₅R^F173L-TG)and hD₅R-wild-type?WT?transgenic(hD₅R^WT-TG)mice.Our previous study only found that hD₅R^F173L-TG mice are hypertensive.²⁴ However,the mechanisms causing the renal D₅R dysfunction in hD₅R^F173L-TG mice are not clear.Our current experiments focused on oxidative stress and urinary sodium metabolism to investigate the causes of increased blood pressure in hD5RF173L transgenic mice.Aims:1.To study the phenotype of blood pressure and urinary sodium excretion in Adn^-/-mice.2.To study the molecular mechanism of reduced natriuresis of adiponectin in SHR.3.The effect of GRK4 on the desensitization of AdipoRs was determined.4.To study the synergistic effect of AdipoR₁ and D₁R and its mechanism.5.The mechanisms of the decreased interact of AdipoR₁ and D₁R in SHR were defined.6.The blood pressure,urinary sodium excretion and other phenotypes of hD₅R^F173L173L transgenic mice were studied.7.The causes of blunted urinary sodium excretion and increased blood pressure in hD₅R^F173L transgenic mice were determined.Methods:1.Phenotypes of blood pressure,urine sodium and urine volume in Adn^-/-mice.?1?A tail-cuff method was used to detected the blood pressure of Adn^-/-mice and their control WT mice.?2?The difference of urinary sodium excretion in Adn^-/-mice and control WT mice was detected by using a mouse metabolic cage.?3?The weights of Adn^-/-and control WT mice were detected with an electronic balance.?4?The genotype of Adn^-/-mice was verified by RT-PCR.?5?The adiponectin expressions of Adn^-/-and WT mice in adipose tissue,plasma and kidney were verified by Western blot.2.The differences of AippoRs expressions and the relationships between AdipoRs and G protein in WKY and SHR.?1?The expressions of adiponectin,AdipoRs and GRK4 in WKY and SHR were detected by western blot.?2?The phosphorylation levels of AdipoRs at the Serine/Threonine residues in WKY and SHR were detected by using the method of immunoprecipitation and immunoblotting.?3?The co-linking of AdipoRs with G?q,G?s,G?i in WKY and SHR were detected by using coimmunoprecipitation.?4?The effect of G?i protein blocker PTX?Pertussis toxin?on adiponectin induced up-regulation of p-AMPK was detected by western blot.?5?The different effects of adiponectin on AMPK activaition in the RPT cells from WKY and SHR were detected by western blot.?6?The different effects of adiponectin on eNOS activaition in the RPT cells from WKY and SHR were detected by western blot.3.GRK4 is the key enzyme for regulating the phosphorylation desensitization of AdipoRs in the kidney.?1?The phosphorylation levels of AdipoRs at the Serine/Threonine residues in GRK4142V transgenic and WT mice were detected by immunoprecipitation and immunoblotting.?2?Intravenous infusion of adiponectin were used to determime the functions of AdipoRs activation in the GRK4 142V transgenic and WT mice.?3?The couplings of AdipoRs with G?i were investigated in the GRK4 142V transgenic and WT mice by coimmunoprecipitation.4.AdipoR₁ and D₁R interact to induce urinary sodium excretion,which is lost in SHRs.?1?Intravenous infusion of fenoldopam or adiponectin was used to determined the effect of D₁R or AdipoR₁ activation on sodium excretion in Adn^-/-or D₁R^-/-mice.?2?Coimmunoprecipitation was used to determine the coupling of D₁R and AdipoR₁ in RPT cells and renal cortices from WKY and SHRs.?3?The change of couplings between AdipoR₁ and D₁R after activation were detected by coimmunoprecipitation in WKY-RPT and SHR-RPT cells.?4?The changes of the co-localization between AdipoR₁ and D₁R after activation on the cell membrane in the RPT cells of WKY and SHR were detected by immunoblotting and laser co-focusing.5.Phenotypes of blood pressure and urinary sodium excretion in hD₅R^F173L173L transgenic mice.?1?A tail-cuff method was used to detected the blood pressure of hD₅R^F173L transgenic and hD₅R^WT transgenic mice at different month ages.?2?The blood pressure of hD₅R^F173L transgenic and mice were also measured by carotid artery intubation under anesthesia.?3?Intravenous infusion of fenoldopam was used to determined the effect of D₅R activation on sodium excretion in the hD₅R^F173L transgenic and hD₅R^WT transgenic mice.?4?The differences of urine sodium excretion in hD₅R^F173L transgenic and hD₅R^WTT transgenic mice at different ages were detected by mouse metabolic cage.6.The role of Trx1 on urinary sodium excretion and blood pressure in D₅R^F173L173L transgenic mice.?1?Western blot was used to detect the differential expression of Trx1 in hD₅R^F173L173L transgenic and hD₅R^WT transgenic mice.?2?Western blot was used to detect the up-regulation of D₅R on Trx1 through PLC/PKC signaling pathway.?3?The association of between D₅R and G?q,G?s were detected by coimmunoprecipitation in hD₅R^F173L transgenic and hD₅R^WT transgenic mice.?4?The phosphorylation levels of D₅R at the sites of serine/threonine in the hD₅R^F173L173L transgenic and hD₅R^WT transgenic mice were detected by the method of immunoprecipitation and immunoblot.?5?Western blot was used to determined the effect of fenoldopam on Trx1 expression in the primary renal proximal tubule cells from hD₅R^F173L transgenic and hD₅R^WT transgenic mice.?6?A PKC activity test kit was used to determined the effect of fenoldopam on PKC activity in the primary renal proximal tubule cells from hD₅R^F173L transgenic and hD₅R^WTT transgenic mice.Results:1.Adn^-/-mice developed hypertension,obesity,accompanied with impaired natriuresis and diuresis without high-salt high-fat induction.2.Compared with WKY,the serum adiponectin concentration was significantly lower in SHR;and the expressions of AdipoRs in the kidney were not statistically different.3.The phosphorylation levels of AdipoRs were increased by 2-3 times at both the serine and threonine residues;and the expression of GRK4 was significantly higher in SHR.4.Our present studes found that the AdipoRs were coupled with G?i,neither G?q nor G?s;and the PTX,an inhibitor of G?i,can block the effect of adiponectin induced activation of AMPK in WKY-RPT cells.5.Knockdown of AdipoR₁ by siRNA blocked the stimulatory effect of adiponectin on AMPK in RPT cells.6.In contrast to WKY,the degree of coupling between AdipoRs and G?i were significantly decreased in SHR;and adiponectin failed to activate AMPK/eNOS signaling pathway in SHR-RPT cells.7.In contrast to WT,the phosphorylation levels of AdipoRs in the kidney from the GK4 142V transgenic mice were significantly enhanced,especially at either the serine residues.8.In contrast to WT mice,the degree of coupling between AdipoR₁ and G?i was significantly decreased in GRK4 142V transgenic mice.9.Intraveous infusion of adiponectin confirmed the impaired natriuresis and dieresis in GRK4 142V transgenic mice.10.The effect of D₁R on urinary sodium excretion was blunted in adiponectin knockout mice;the effect of of AdipoR on urinary sodium excretion was almost lost in D₁R knockout mice.11.There was an association between D₁R and AdipoR₁;the degree of coupling was significantly reduced in SHR,in contrast to WKY;stimulation of one receptor can enhance the co-linking of the other receptor with it,vice versa,in WKY-RPT cells,not in SHR-RPT cells.12.Activation of D₁R or AdipoR₁ could increase the membrane expression of another receptor,vice versa,in WKY-RPT cells,not in SHR-RPT cells.13.D₁R and AdipoR₁ are co-located on the cell membrane;activation of D₁R or AdipoR₁ could increase the co-localization in WKY–RPT cells,not in SHR-RPT cells.14.Compared with WT,the blood pressure of hD₅R^F173L transgenic mice was significantly increased,acompanied with impaired urinary sodium excretion.15.Intranveous perfusion experiment showed that,compared with WT,the effect of fenoldopam on urinary sodium excretion was significantly reduced in the hD₅R^F173L173L transgenic mice.16.Activation of D₅R specifically upregulated the expression of Trx1 in the RPT cells via PLC/PKC signaling pathway.17.The phosphorylation levels of D₅R at serine/threonine residues were significantly increased in the renal cortices from the hD₅R^F173L transgenic mice,compared to hD₅R^WTT transgenic mice.18.The degree of couplings of D₅R with G?q and G?s were significantly lower in the kidney of the hD₅R^F173L transgenic mice than in hD₅R^WT transgenic mice.19.Fenoldopam failed to upregulate the expression of Trx1 in primary cultured RPT cells from hD₅R^F173L transgenic mice.20.In contrast to hD₅R^WT transgenic mice,the stimulatory effect of fenoldopam on the PKC activity was absent in the primary cultured RPT cells from hD₅R^F173L173L transgenic mice.Conclusions:1.Adn^-/-mice spontaneously developed high blood pressure and accompanied with impaired urinary sodium excretion.2.Adiponectin induced natriuresis and diuresis through AdipoR₁/AMPK/eNOS signaling pathway.3.Adiponectin receptor has physiological coupling with G protein,and it is a new type of G protein coupled receptor.4.The increased GRK4 activity in SHRs,by hyper-phosphorylated AdipoRs,impaired its natriuresis and diuresis,involved in the pathogenesis of hypertension.5.AdipoR₁ and D₁R interacted to induce natriuresis and dieresis;the lost synergy of them involved in the pathogenesis of hypertension.6.The increased blood pressure and oxidative in the hD₅R^F173L transgenic mice stress were associated with hyperphosphorylated D₅R,which led to disassociation with G?q and G?s,and failed to upregulate Trx1 via PLC/PKC signal pathway.