Angiotensin 1-7 administration alters baroreflex sensitivity and renal function in sympathectomized rats

Background: Renin-angiotensin system (RAS) plays an important role in cardiovascular and kidney functions to regulate the systematic blood pressure. The baroreflex sensitivity (BRS) index is a quantitative index that was considered as a prognostic indicator for cardiovascular system risk which is known as heart rate change to blood pressure change ratio ( HR MAP ∆ ∆ ). Sympathetic nerve is an arm of the BRS which may be influenced by RAS vasodilatory arm function. Objectives: To determine the role of angiotensin 1-7 (Ang1-7) accompanied by bilateral renal denervation (RDN) on BRS and renal function. Methods: Male and female anesthetized Wistar rats were subjected to RDN and treated with Ang1-7 (300 and 1000 ng.kg-1.min-1). Mean arterial pressure (MAP) and heart rate (HR) responses to phenylephrine (PE) infusion were measured to determine BRS index. As kidney function markers, the creatinine clearance (CrCl) and urine flow (UF) were also determined. Results: The results showed that BRS increased significantly in RDN male rats treated with Ang1-7 compared to vehicle (P<0.05). BRS attenuates significantly in RDN male rats treated with vehicle or Ang1-7 when compared with non-RDN (control). In female rats, the BRS decreased significantly in Ang1-7 treated group (P<0.05). Also, the BRS was significantly different between non-RDN and RDN female rats treated with Ang1-7 (P<0.05). The ClCr in female and UF in both genders increased by Ang1-7 infusion in RDN and non-RDN rats. The alteration of serum nitrite level by Ang1-7 in non-RDN and RDN groups was gender related. Conclusion: The Ang1-7 infusion could alter the BRS index in RDN rat’s gender dependently. The ClCr response to Ang1-7 infusion in male rats was dose related.


Background
One of the main arms of renin-angiotensin system (RAS) that act against angiotensin II (AngII) is angiotensin 1-7 (Ang1-7) (1).One of the homeostatic factors that interact with RAS is baroreceptor reflex sensitivity (BRS) index.BRS in known as heart rate change to blood pressure change ratio ( HR MAP ∆ ∆ ) which represents the level of baroreflex activity, vascular tone and vascular resistance (2).The BRS as a prognostic indicator for cardiovascular system risk could be altered by angiotensin II (AngII) infusion in the brain (3).However the results would be different if AngII infused systematically.Ang1-7 is one of the vasodilatory arm of RAS that not able to cross the blood-brain barrier, and it seems that the role peripheral Ang1-7 on the BRS index and renal function needs to be studied.In addition, the Ang1-7 receptor is reported to be acted gender related (4).Therefore we hypothesized that Ang1-7 infusion alters the BRS index and renal function, and this hypothesis was tested in bilateral renal denervation (RDN) and non-RDN male and female animal's model.

Objectives
To determine the role of Ang1-7 infusion accompanied by bilateral RDN on BRS and renal function in rats.

Experimental Surgery 1. Surgical preparation
The rats were anesthetized with urethane (1.7 g.kg -1 body weight, Sigma St. Louis USA), and the trachea was cannulated.In order to measure mean arterial pressure (MAP) and heart rate (HR), the left carotid artery was cannulated linked to a transducer cable to Quad Bridge Amp (ADINSTRUMENTS, model: ML224, S/N: 224-0265, Australia) connected to PowerLab 4/30 hardware (ADINSTRUMENTS, model: ML866, S/N: 430-0658, Australia) and LabChart software (ADINSTRUMENTS, v7.3.7,Australia).The RDN was performed by sectioning the left and right renal sympathetic nerves.The renal sympathetic nerves were similarly exposed and manipulated without RDN in non-RDN groups.The rectal temperature was controlled at 37± 2°C, and the bladder was cannulated for urine collection..min-1 ) were administrated via jugular vein using micro-syringe pump (New Era Pump System Inc. Farmingdale, NY, USA).Fifteen minutes post saline or Ang1-7 infusion, MAP and HR responses to saline or Ang1-7 infusion were measured over the last 5 minutes of infusion (c) Response to PE data; the single bolus dose of α-adrenergic receptor agonist, PE (0.1 mg.kg -1 body weight) (5) was injected intravenously via the jugular vein.MAP and HR changes (ΔMAP and ΔHR) by the peak amplitude of pressure and bradycardia responses were measured.At the end of PE infusion, the collected urine volume and blood sample were obtained.The levels of serum and urine Cr using quantitative diagnostic kits (Pars Azmoon, Iran) and the serum level of nitrite by the Griess method were measured.The Cr clearance (ClCr) was calculated by clearance formula as follows: ClCr=Urine flow(UF)*Urine Cr level/ Serum Cr level.

Ethical issues
The experimental protocol was approved by Isfahan University of Medical Sciences Ethics Committee (Code # ir.mui.rec.1395.3.811).Prior to the experiment, the protocols were confirmed to be in accordance with the guidelines of Animal Ethics Committee of Isfahan University of Medical Sciences.

Statistical analysis
The data is reported as the mean ± SEM.Repeated measures ANOVA for MAP and HR and one-way ANOVA for ClCr, UF and serum nitrite levels were employed to compare between the groups using Tukey test as post hoc test.The Student t test was applied to find the statistical difference between the genders and P ≤0.05 was considered to be significant.

Discussion
It was reported that renal sympathetic nerves and RAS have reciprocal interaction (6).RAS components alter the renal sympathetic nerve activity (RSNA) (7).It was reported that RSNA can be altered by intravertebral injection of AngII (8).Also, it has been shown that infusion of AngII in the brain alters the baroreflex activity and RNSA (9).RDN resets or restores the baroreflex control of RSNA and reduces blood pressure in resistant hypertension (10,11).The main findings of our study indicated that the BRS increased in RDN male rats treated with Ang1-7 compared to vehicle or Ang1-7.Also, the BRS increased in non-RDN male rats compared to RDN when treated with Ang1-7 in a similar manner.Different results of BRS in different gender may be related to sex hormone as it has been shown that estrogens can modulate the BRS in normotensive rats (12,13).Also, it is possible that a lesser sympathetic output to the kidney of females may contribute to their lower blood pressure (14).RDN reduces hypertension in male SHR rats (15,16).Chen and DiCarlo reported that the performance and efficiency of baroreflex function in female were more than male rats (17).In line with our result, it is possible that elevated nitric oxide (NO) signaling increases after radio-frequency RDN and plays a role in the reduction in MAP (18).Finally, the effect of Ang1-7 on ClCr and UF seems to be related to the ) and the interaction between group treatment and time (P group x time ).
(a) Baseline; after 30 minutes of equilibration, the baseline data (the last 5 minutes) for MAP and HR were determined.(b) Treat with Ang1-7; after equilibrium time, saline or Ang1-7 (300 or 1000 ng.kg -1

Table 1 .
Data for baroreflex sensitivity (BRS) index, creatinine clearance (ClCr), urine flow (UF) and serum nitrite level (Nitrite) in intact (non-RDN) and renal denervation (RDN) male and female rats in the groups treated with vehicle or two different doses of Ang1-7 infusion Data for mean arterial pressure (MAP) and heart rate (HR) in intact (non-RDN) and renal denervation (RDN) rats in equilibrium state (Control), vehicle/Ang1-7 (300/1000 ng.kg -1 .min - ) infusion (Treat), and phenylephrine infusion (Phe).ANOVA for repeated measure was applied to compare physiological parameters using factor group treatment (Pgroup), time (Ptime) and the interaction between group treatment and time (Pgroup x time) Data for mean arterial pressure (MAP) and heart rate (HR) in intact (non-RDN) and renal denervation (RDN) rats in equilibrium state (Control),