G Protein-coupled Receptor Kinases 4 Impaired Adiponectin Receptor-induced Renal Sodium Excretion In Hypertension

BackgroundThe impaired natriuresis is the distinguishing feature of essential hypertension.However,the machanism is not clear.As the essential endocrine organ,the adipose tissue had a relationship with renal function.Adipocytes secrete numerous hormones,the adipocytokines mediate crosstalk among different cell populations within adipose tissue and also travel to remote organs to regulate systemic energy metabolism.The level and action of adipocytokines are often altered.In pathological situations there is a change in the profile of adipokines released,with less production of the “healthy” adipokines such as adiponectin and more release of the “unhealthy” adipokines,leading to the development of cardiovascular disease,especially hypertension.In various adipocytokines,adiponectin is the most significant one,it is a great amount of secretion hormones mainly produced by white adipose tissue.The relationship of adiponectin and hypertension is identified,decreased plasma adiponectin concentration is in patients with essential hypertension.Salt-induced hypertension is happened in adiponectin knockout mice.Adiponectin supplementation ameliorates hypertension of genetically obese mice.However,the mechanism of adiponectin in the regulation of blood pressue(BP)is not clear.The effect of adiponectin is mainly via adiponectin recepors.They are classified into two types: Adipo R1 and AdipoR2.Adiponetin receptors are widely distributed,including kidney.Our initial studies also found that adiponectin receptors expressed in kidney,especially in renal proximal tubular(RPT)cells.It is possibly that adiponectin,may via adiponectin receptors,in the regulation of renal sodium excretion.However,the effect was impaired in hypertension,the mechanism need further explored.AdipoR belongs to the progestin and adipo Q receptor groups(PARQs),which is intergral membrane protein that contain seven membrane spanning,similar with G protein coupled receptors(GPCRs),and it has been reported AdipoR phosphorylated by G ptotein coulpled receptors kinases(GRK).The impaired action of Adipo R many due to the phosphorylation,and involved in the development of hypertension.According to numerious provious studies,The wide variety of GPCRs that are responsible for optimal BP control leaves no doubt that GRKs play a vital role in the regulation of BP.In the regulation of renal sodium excretion,GRK4 is the most important GRKs,it plays an essential physiological role in the long-term control of BP and in sodium homoeostasis via phosphorylation of the renal dopamine receptors,and up regulation of Angiotensin II type 1 receptor.Human GRK4 gene locus in chromosome 4p16.3 which is related to essential hypertension.Three missense single nucleotide polymorphisms(SNPs ie,65R>L,142A>V,and 486A>V)in the coding region of GRK4 are associated with increased blood pressure.A number of studies have shown the genetic association of the 3 GRK4 SNPs with human essential hypertension in several ethnic groups.Using GRK4,the accuracy of prediction of essential hypertension is 70% in African Ghana.In a Japanese cohort,the presence of all 3 GRK4 variants impaired the natriuretic effect of a dopaminergic agonist and correctly predicted the presence of saltsensitive hypertension in 94% of cases.Our meta analysis of multicenter of experiments reveal GRK4 SNPs are associated with susceptibility of essential hypertension.BP in transgenetic mices of 3 human GRK4 SNPs are increased,especially GRK4? 142 V transgenetic mice,they are hypertensive even fed a normal salt diet.GRK4 activity is increased in the kidneys of humans with essential hypertension.Therefore,we deduced that GRK4 may participate in the regulation of renal Adipo R phosphorylation and play a role in the pathogenesis of hypertension.Depletion of renal GRK4 increases sodium excretion and urine volume and lowers BP in spontaneously hypertensive rat(SHR).Therefore,suppression of GRK4 in the kidney is an important issue and might be an alternative method for lowering BP in hypertension.In conclusion,We hopothesis that adiponecin,via adiponectin receptors,play an important role in the regulation of sodium excretion,the impaired adiponectin-induced sodium excretion might be involved in hypertension.GRK4 may participate in the phosphorylation of adiponectin receptors and contributed to the impaired function of adiponectin.To explored a new targeted therapy to decrease the renal expression of GRK4 is essential in the treatment of hypertension.Objective:To identify the role of adiponectin and adiponectin receptor in the regulation of renal sodium excretion,and the effect in the pathogenesis of hypertension.Second,to find the relationship of GRK4 and adiponectin receptor phosphorylation and densensititation in hypertension.Third,Whether UTMD-GRK4 si RNA in SHR could have an effect on sodium excretion and BP via reducing adiponectin receptor phosphorylation.Methods:1.The effect of adiponectin in the regulation of sodium excretion1.1 Determine the expression of Adiponectin receptors in renal and RPT cells by immunofluorescence and immunoblotting.1.2 Intrarenal infusion of adiponectin in WKY rats to make sure whether adiponectin induced natriuresis and diuresis by observing the urine flow(UV),absolute sodium excretion,and the BP during infusion.To exclude the osmotic diuresis or allergenic effect caused by protein,boiled adiponectin was also infused in the same way.1.3 To determine the effect of adiponectin on Na+-K+-ATPase activity in WKY RPT cells.1.4 Transfecting AdipoR1 siRNA and Adipo R2 si RNA in WKY RPT cells,to understand which receptor(s)are involved in the regulation of Na+-K+-ATPase activity in WKY RPT cells.1.5 Using the inhibitor of(Adenosine 5‘-monophosphate-activated protein kinase)AMPK?Endothelial nitric oxide synthase(e NOS),to find out the signal transduction in the inhibitory effect of adiponectin on Na+-K+-ATPase activity,and immunobloting to determine the level of phosphorylation of AMPK and e NOS after stimulated by adiponectin.2.The natriuretic effect of adiponectin in SHR2.1 Intrarenal infusion of the same dose of adiponectin in SHR to know the natriuretic effect of adiponecin in hypertension,the urine flow and absolute sodium excretion,and the BP.2.2 To observe the effect of adiponectin on Na+-K+ATPase activity in SHR RPT cells.2.3 Realt-time PCR to determine the mRNA expression of adiponectin receptors in SHR RPT cells,and in the mean while,to find out the protein expression of adiponectin receptors in SHR RPT cells.2.4 Intrarenal infusion of the activator of adiponectin receptor Adipo Ron in SHR to futher determine the impaired natriuretic effect of adiponectin in SHR is due to the impaired receptors.2.5 Depending upon the results,the plasmid of AdipoR1 and AdipoR2 were designed to increase the expression of adipinectin receptors,and then to research after overexpression of Adipo R1 and Adipo R2,whether the inhibitory effect of adiponectin on Na+-K+ATPase activity in SHR RPT cells would be restored.2.6 Using the co-immunoprecipitation to observe the phosphorylation of adiponectin receptors in SHR RPT cells?2.7 Intrarenal infusion of AdipoRon in GRK4?142V transgenetic mice,to observe whether the natriuretic effect of adiponectin receptor is impaired in GRK4?142V transgenetic mice,and to understand whether GRK4 is involved in the regulation of adiponectin receptors.2.8 Real-time PCR and immunobloting to choose the most efficient GRK4 siRNA,and then transfected GRK4 siRNA into SHR RPT cells,and then research the phosphorylation of adiponectin recrptors.3.Testing the urine sodium excretion and BP after Ultrasound-targeted microbubble destruction(UTMD)-GRK4 siRNA delivery in SHR and the involved mechanism.3.1 Preparation of the Microbubbles Carrying GRK4 si RNA.Fluorescence microscopy confirmed the effective of microbubbles conjugated with the 5-FAM-labeled GRK4 si RNA.3.2 Check the renal GRK4 expression after UTMD-GRK4 si RNA delivery in SHR,and measure the BP,24 h urine flow,24 h urine absolute sodium excretion,renal function,and renal fibirosis.3.3 To discover the phosphorylation of adiponectin receptor after UTMD-GRK4 siRNA delivery.Result1.Both Adipo R1 and AdipoR2 were expression in kidney,especially in the cortex,and renal proximal tubules.The specific AdipoR1 protein(?43 k Da)band and Adipo R2 protein(?44 kDa)band in kidney.2.Vary dose of adiponectin increased urine flow(UV)and absolute sodium excretion from(UNaV)while the BP had no change,suggesting that the natriuresis and diuresis caused by adiponectin is not the consequence of change in BP.3.Adiponectin inhibited the Na+-K+-ATPase activity in a concentration-and timedependent manner in RPT cells from WKY rats.4.After inhibition of mRNA and protein expression of Adipo R1 and AdipoR2 by siRNAs,the inhibitory effect of adiponectin on Na+-K+-ATPase activity was completely blocked,indicating the specificity of adiponectin as an adiponectin receptor agonist.5.The inhibitory effect of adiponectin on Na+-K+-ATPase in RPT cells is via AMPK-e NOS signal way.In the presence of AMPK inhibitor compound C or e NOS inhibitor L-NAME(NG-nitro-l-arginine methyl ester),the inhibitory effect of adiponectin was lost.The involvement of AMPK and e NOS was also confirmed by our further experiment.Stimulation with adiponectin increased phosphorylation of AMPK in a time dependent manner;similarly,adiponectin increased phosphorylation of e NOS,while in the presence of compound C,the stimulatory effect of adiponectin on eNOS phosphorylation was blocked;indicating AMPK is the upstream signal of eNOS.6.Infused adiponectin into SHR via supra-renal artery,it resulted that adiponectin did not induce natriureis and diuresis until the dosage of 5?g/kg,indicating the impaired adiponectin-mediated natriuresis in hypertension.Similarly,the inhibitory effect of adiponectin on Na+-K+-ATPase activity was reduced in RPT cells from SHR,comparing with WKY,the inhibitory effect of adiponectin began to attenuated at the point of 50ng/ml in SHR.The effect of adipo Ron induced natriuresis and dieresis is also impaired in SHR.7.The reduced adiponectin-induced natriureis and diuresis might,at least in part,be accounted by reduced adiponectin receptor expression,because we found that both AdipoR1 and AdipoR2 receptor m RNA and protein expressions were lower in kidneys from SHR as compared with WKY rats.However,it should be noticed that besides of decreased receptor expression,there might be other factors affecting the effect of adiponectin receptor function,after overexpression of AdipoR1 and Adipo R2 receptor by plasmids,the inhibitory effect of adiponectin on Na+-K+-ATPase activity was only partially reversed in RPT cells from SHR.8.We found that AdipoR1 and AdipoR2 were phosphorylated,which might be the result of impaired action of adiponectin.9.To test wehther GRK4 is the cause of phosphorylation of Adipo R in SHR RPT cells,we infused AdipoRon in GRK4?142V trans genetic mice.It resulted that comparingwith GRK4?WT mice,the adiponectin receptor mediated natriureis and dieresis effect were lost.After tranfected GRK4 siRNA to SHR RPT cells,the phosphorylation of adiponectin receptor were improved,which indicated that GRK4 participated the regulation of AdipoR phosphrylation.10.In SHR,UTMD-targetedGRK4 si RNA delivery to the kidney reduces GRK4 expression,improved adiponectin receptors phosphorylation and consequently increased sodium excretion,and leads to lowered BP.Although the renal fibrosis is significantly different between SHR and the UTMD-targeted GRK4 siRNA–treated SHR,the renal function,determined by BUN and creatinine,has no difference between two groups.This suggests that UTMD-mediated siRNA delivery is an efficient method for the delivery of siRNAs to the kidney and a potential clinical strategy for lowering BP in the future.Conclusion:Adiponectin,as an important link between adipose tissue and kidney,play an important role in natriuresis and diuresis.The impaired function of adiponectin on renal sodium excretion is not only due to the decreased expression of adipionrctin receprors,more importantly,the phosphorylation of Adipo R which regulated by increased activities of GRK4.UTMD delivery of GRK4 si RNA to SHR kidney reduces GRK4 expression,improved adiponectin receptors phosphorylation and associated sodium excretion,and leads to lowered BP,indicate that UTMD-mediated GRK4 si RNA delivery may provide a promising novel strategy for gene therapy for hypertension.