Irisin Promoted Urinary Sodium Excretion By Regulating Renal G Protein-coupled Receptor Kinases 4 And Its Effects On Lowing Hypertension

Objective: Hypertension is the most important risk factor for cardiovascular disease(CVD).Exercise intervention strategy is considered one of the most effective non-drug therapies in most major guidelines for hypertension.Irisin,a recently discovered myokine,has been shown to mediate multiple regulatory effects of skeletal muscle on body homeostasis.It remains unclear whether irisin can play a role in the long-term regulation of hypertension.In this study,we investigated the long-term effects of irisin on blood pressure and renal urinary sodium excretion.Methods: 1.Adult C57BL/6 mice(WT)and irisin knockout mice,ranging in age from 8-12 weeks,were used to establish the exercise model of hypertensive mice.The experimental period was 8 weeks,and the two kinds of mice were divided into3 groups respectively: 1)control group: mice did not participate in exercise training,and were given continuous subcutaneous infusion of saline with ALZET osmotic pumps from the 5th week of the experimental period;2)sedentary group: mice did not participate in exercise training,and were given continuous subcutaneous infusion of Ang-II(400ng/kg/min)with ALZET osmotic pumps from the 5th week of the experimental period;3)exercise group: mice were subjected to 8 weeks of aerobic training on a treadmill(0% incline and speed of 12 m/min,lasting 60min/day,five times a week)and given continuous subcutaneous infusion of Ang-II(400ng/kg/min)with ALZET osmotic pumps from the 5th week of the experimental period.Systolic blood pressure(SBP),diastolic blood pressure(DBP),urine volume and urinary sodium were monitored weekly to observe the changes in hypotensive effects of exercise after irisin knockout.2.Adult WKY rats and SHRs,ranging in age from 8-12 weeks,were used to establish the irisin treatment model.The experimental period was 4 weeks,and the rats weredivided into 4 groups: 1)WKY control group: rats were given continuous subcutaneous infusion of saline with ALZET osmotic pumps;2)WKY+irisin treatment group: rats were given continuous subcutaneous infusion of irisin(1μg/kg/day)with ALZET osmotic pumps;3)SHR control group: rats were given continuous subcutaneous infusion of saline with ALZET osmotic pumps;2)SHR+irisin treatment group: rats were given continuous subcutaneous infusion of irisin(1μg/kg/day)with ALZET osmotic pumps.SBP,DBP,urine volume and urinary sodium were monitored weekly to observe the effect of long-term exogenous irisin treatment on hypertension,glucose and lipid metabolism.3.Adult C57BL/6 mice and irisin overexpression(OE)mice,ranging in age from 8-12 weeks,were used to establish Ang-II induced hypertension model.The experimental period was 4 weeks,and the mice were divided into two groups: 1)WT+Ang-II group: mice were given continuous subcutaneous infusion of Ang-II(400ng/kg/min)with ALZET osmotic pumps;3)irisin-OE+Ang-II group: irisin-OE mice were given continuous subcutaneous infusion of Ang-II(400ng/kg/min)with ALZET osmotic pumps.SBP,DBP,urine volume and urinary sodium were monitored weekly to observe the effect of endogenous irisin overexpression on hypertension.Results: 1.Long-term exercise training significantly increased the urine volume and urinary sodium excretion in hypertensive models of C57BL/6 mice,and reduced their SBP and DBP.The above antihypertensive effect of exercise training was significantly weaken in hypertensive models of irisin knockout mice.2.Long-term subcutaneous infusion of exogenous irisin significantly increased urine volume and sodium excretion of SHR rats,reduced their SBP and DBP,and had no significant effect on the blood pressure of WKY rats.Meanwhile,it showed a tendency to improve glucose and lipid metabolism in both WKY rats and SHRs.3.Endogenous irisin overexpression could antagonize Ang-II-induced hypertension,increase urine volume and sodium excretion in hypertensive mice,and reduce their SBP and DBP.Conclusion: Irisin can promote renal urinary sodium excretion,thereby reduce high blood pressure in hypertensive rats and mice,but show no significant effect on normal blood pressure.Objective: The kidney plays a central role in the long-term regulation of arterial blood pressure through urinary sodium excretion.Renal dopamine receptor dysfunction leads to urinary sodium excretion disorder and increased blood pressure.The increased activity of G protein coupled receptor kinase 4(GRK4)is an important cause of dopamine receptor dysfunction in hypertension.Previous studies have shown that exercise training can improve the activity of dopamine D1 receptor in the kidney of elderly rats and promote urinary sodium excretion.The purpose of this study was to investigate the effects of the myokine irisin on renal GRK4 expression and urinary sodium excretion.Methods: 1.Intrarenal infusion of the activator of D1 receptor fenodopam was performed in the WKY rats and SHRs irisin treatment model.Urine flow and urine sodium excretion rate were monitored to evaluate the changes of renal dopamine receptor function.Western blot was used to detect the phosphorylation level of renal D1 receptor among different groups.2.Real-time quantitative PCR and western blot were used to compare the expression levels of renal GRK4 in the WKY rats and SHRs treated with irisin or saline.3.Immoralizable RPT cell lines from WKY and SHR rats were preincubated with irisin(1μg/ml)or PBS for 24 h.Real-time quantitative PCR and western blot were used to compare the expression levels of GRK4 among the different RPT groups.Results: 1.Intrarenal infusion of fenoldopam via the suprarenal artery significantly increased urine flow and sodium excretion in a dose-dependent manner in WKY control group,WKY+irisin treatment group,SHR+irisin treatment group,but not in SHR control group.However,there was no significant difference between WKY+irisin treatment group and WKY control group.Western blotting showed that irisin treatment significantly reduced the phosphorylation level of dopamine D1 receptor in SHR rats,but had no significant effect on WKY rats.2.Irisin treatment significantly downregulated the m RNA and protein expression of renal GRK4 in SHR but not in WKY rats.3.Irisin preincubation significantly downregulated the m RNA and protein expression of GRK4 in immoralization SHR-RPT but not in WKY-RPT.Conclusion: Irisin can improve the function of dopamine receptors in the kidney,and promote urinary sodium excretion by down-regulating renal GRK4 expression.Objective: The role of G protein coupled receptor kinase 4(GRK4)in the occurrence and development of essential hypertension has been paid much attention.G protein–coupled receptor kinase 4(GRK4)is involved in activity of dopamine receptor D1 in renal proximal tubules and thus mediates sodium reabsorption and blood pressure(BP)regulation.Our previous experimental results showed that irisin could downregulate the expression of renal GRK4 both at m RNA and protein levels.Therefore,the transcriptional regulation of GRK4 is the focus of our research.In this study,we investigated the underlying mechanism of irisin on downregulation of GRK4 expression at the transcriptional level.Methods: 1.Immoralizable RPT cell lines from WKY and SHR rats were preincubated with irisin(1μg/ml)or PBS for 24 h.Immunofluorescence staining,nuclear and cytoplasmic extraction,and western blot were used to compare the intracellular distribution of irisin among the different RPT groups.2.Immunofluorescence staining,nuclear and cytoplasmic extraction,and western blot were used to compare the intracellular distribution of irisin in the kidney of the WKY rats and SHRs irisin treatment model.3.The GRK4 reporter gene vector was constructed in HEK293 T cells;The GRK4 promoter activity was evaluated by dual luciferase reporter assay when co-transfected with irisin overexpression plasmid.4.Immoralizable RPT cell lines from WKY and SHR rats were preincubated with irisin(1μg/ml)or PBS for 24 h.Chromatin immunoprecipitation(Ch IP)assay was performed to evaluate the abundance of c-Myc-bound GRK4 promoter.Results: 1.Immunofluorescence staining,nuclear and cytoplasmic extraction,and western blot showed that irisin increased in the nucleus of renal cortex in SHRs after treatment with irisin,but not significantly increased in the nucleus of renal cortex in WKY rats.2.Immunofluorescence staining,nuclear and cytoplasmic extraction,and western blot showed that irisin significantly increased in the nucleus of SHR-RPT after incubation with irisin,but no significantly increased in WKY-RPT.3.Dual luciferase reporter assay found that irisin had a weak promoting effect on the transcription of GRK4,suggesting that irisin could not directly downregulate the transcription of GRK4.4.CHIP-PCR showed that irisin significantly decreased the abundance of c-Myc-bound GRK4 promoter,suggesting that irisin could down-regulate the expression of GRK4 at the transcriptional level by affecting the binding of c-myc to GRK4 promoter.Conclusion: Irisin could downregulate the expression of GRK4 at the transcriptional level by affecting the binding of c-myc to GRK4 promoter.