Role Of GRK4 Variant A142V In The Regulation Of Renal ETB Receptor In Hypertension

Background:Hypertension is an significant globle health problem.Patients with hypertension are at an increased risk for heart failure(HF),stroke, renal disease and acute myocardial infarction. However, the pathogenesis of hypertension are poorly understood, Sodium and water excretion dysfunction had be proved and accepted to play an critical role in the regulation of blood preasure, kidney has unlimited potential to regulate long-term regulation of blood volume and pressure.. Endothelin-1(ET-1),a potent endothelial derived pressor peptide, which also excessively secreted from renal turbule cells has been suggested to play a significant contribution in the regulation of water and sodium excretion, Renal endothelin system is including two receptors, Endothelin receptor type A(ETAR) and Endothelin receptor type B(ETBR),they had different distribution,structure and pharmacological characteristics. ETB receptor, not ETA receptor, abundantly expresses in renal proximal tubule. affect the reabsorption of sodium and water. Studies found that activition of ETBR can regulate sodium transport in the renal proximal tubule, in vivo directly and also, by interacting with the other receptors such as dopamine receptor and angiotension II type 1(AT1) receptor In our previous study we found ETBR’s expression and function were impaired in Spontaneously hypertensive rat, and the aberrant interaction with other receptors are involved in the development of hypertension. However, the mechanism of impairment of ETBR remind to be clarified.ETBR belongs to the G protein coupled receptors, like other GPCRs, ETBR’s expression and function were modified by phosphorylation and dephosphorylation.It is widely accepted that increased GRK activity is crucial in regulation of phosphorylation of GPCR in Hypertension. The GRK family is including 7 members. The limited expression of GRK4 and the fact that the GRK4 gene locus linked to hypertension make the GRK4 gene an attractive candidate for a genetic determinant to human essential hypertension. Our previous studies found that the GRK4 and its SNPs including 65 L,486V,142 V are associated with hypertension in various people including Chinese, American,Africans, Australians and Italian. Human Constitutively activtion of GRK4 variants could increase the activity of GRK4.Mice overexpressing Human GRK4 A142 V are hypertensive even on a normal NaCl intake. In contrast, human GRK4 A486 V transgenic mice, depending on the genetic background, become hypertensive only after an increase in sodium intake. In our previous study we found the blood pressure of GRK4 A142 V transgenic mice were higher than wild type, companied with urinary and sodium excretion dysfunction, by downregulating renal D(1) receptor expressions and impaired natriuretic effect. And GRK4 A142 V variants can increase vascular AT1 receptor expression, increased Ang II-induced vasoconstriction, there by increasing blood pressure. However, those are still can not fully explain the closely association of GRK4 A142 V variants and hypertension,And there were no immediate reports about whether ETBR was rugulatated by GRK4.We hypothesis that ETBR was regulated by GRK4,the variant A142 V of GRK4 is the cause of aberrant ETBR, and the impaird natriuretic effect of ETBR may play an important role in hypertension.To verify the hypotheses, we studied the natriuretic effect of ETBR,and the role of GRK4 variant A142 V in the regulation of renal ETB receptor in hypertension.Methods:1 Monitoring Arterial blood pressure(BP) of SHR and WKY rats in basic condition. The diuresis and natriuresis effect of ETBR were measured by adrenal artery perfusion. The mRNA expression of GRK4 and ETBR was analysised by QT-PCR while the protein expression of GKR4 and ETBR was tested by Western blot.2 Comparing the Na+-K+-ATPase activity of WKY and SHR RPT cells,then test the Na+-K+-ATPase activity of RPT cells stimulated of ETBR agonists BQ3020. To study the Na+-K+-ATPase activity of SHR RPT cells which transfected GRK4 siRNA,and then measure Na+-K+-ATPase activity in the effect of ETBR agonists BQ3020.3 Basal measurement including the arterial blood pressure(BP) 、 24-hour urine volume,24-hour urinary sodium of GRK4 A142 V and GRK4 WT transgenic mice,the diuresis and natriuresis effect of ETBR on those mice were measured by jugular vein perfusion of ETBR agonists BQ3020.4.Comparing the Na+-K+-ATPase activity of mouse RPT cells transfected with GRK4 A142 V and GRK4 WT plasmid and then test the Na+-K+-ATPase activity of RPT cells stimulated of ETBR agonists BQ3020.5 Laser confocal was employed to reveal the location of GRK4 and ETBR, The interaction between GRK4 and ETBR were verified with immune co-precipitation.Result:1.The blood pressure of SHRs are higher than WKY rats. The diuresis and natriuresis effect of ETBR was impaired in SHRs. There’s no significant different protein expression of renal ETBR between SHR and WKY, but the expression of GRK4 and the phosphorylation of ETBR was increased in SHR. The inhibitory effect of ETBR on Na+-K+-ATPase activity of WKY renal proximal tubule cells was impaired in SHR renal proximal tubule cells,and can be recovered when transfected GRK4 siRNA.2.The blood preasure of GRK4 A142 V transgenic mice blood is increased and the ETBR-mediated diuresis and natriuresis was impaired compared with Wild type.The inhibitory effect of ETBR on Na+-K+-ATPase activity of mouse renal proximal tubule cells transfected GRK4 A142 V plasmid was impaired.3. The GRK4 and ETBR were co-localization in renal proximal tubuler cells, and this may be the mechanism of the impaired expression and function of ETBR in hypertension.Conclusion:GRK4 variants A142 V regulate ETBR by direct physical receptor interaction, the increased activity of GRK4 mediation of the impaired natriuretic effect of ETBR may participate in the pathogenesis of high blood preasure in the SHR.