Effects of renal sympathetic denervation using saline-irrigated radiofrequency ablation catheter on the activity of the renin-angiotensin system and endothelin-1

Abstract

Introduction:

Excessive activation of the sympathetic nervous system (SNS) and the renin-angiotensin system (RAS) are crucial and interacted closely in the pathogenesis of chronic cardiovascular diseases. This study investigated the effects of renal denervation (RDN) on the RAS.

Materials and methods:

Eight Chinese Kunming dogs underwent bilateral RDN utilizing saline-irrigated radiofrequency ablation catheter. Blood pressure (BP) measurements, blood sampling assays and renal angiography were performed at baseline, 30 min, one month and three months after ablation.

Results:

During three months of follow-up, RDN caused a significant and uniform reduction in plasma level of renin, angiotensin II, and endothelin-1(ET-1), with the reduction of –5.7±6.8 (p=0.049), –19.4±19.3 (p=0.025), and –22.4±21 pg/ml (p=0.02) for plasma renin, –10.6±7.2 (p=0.004), –15.9±8.8 (p=0.001), and –15.2±9.6 pg/ml (p=0.003) for plasma angiotensin II, as well as –3.9±3 (p=0.007), –10.8±5 (p<0.001), and –14.6±6.1 pg/ml (p<0.001) for plasma ET-1. RDN utilizing a saline-irrigated catheter also caused a progressive and substantial BP reduction of –19±22/–8±13, –30±13/–13±14, and –36±20/–16±14 mm Hg (p=0.045, p<0.001, and p<0.002 for systolic BP; p=0.14, p=0.036, and p=0.014 for diastolic BP) without ablation-related complications.

Conclusion:

RDN substantially decreased BP and also significantly decreased the plasma levels of RAS and ET-1, which might be implicated in the mechanism of BP reduction by RDN.

Keywords

Renal sympathetic denervation renin-angiotensin system endothelin-1 renal artery ablation saline-irrigated radiofrequency ablation catheter

Introduction

Excessive sympathetic drive is undoubtedly a major contributing factor to the pathophysiology of hypertension,¹ heart failure,^2,3 end-stage renal disease,^4,5 and many other chronic cardiovascular diseases.⁶ Additionally, the critical role of the circulating renin-angiotensin system (RAS), which is activated by plasma renin, in the regulation of blood pressure (BP) and sodium homeostasis has been recognized for many years. Much of the excessive sympathetic drive in these conditions is directed to the kidney, where it leads to inappropriate sodium retention, renin stimulation, and diminished renal function.⁷ Accumulating evidence showed that the sympathetic nervous system (SNS) and RAS were overexcited and interacted in cardiovascular pathophysiological conditions mentioned above.⁸ Therefore, treatment of the elevated SNS and RAS activity would be anticipated to provide significant clinical benefit.

Recently, renal denervation (RDN) using a catheter-based approach, which targets at disrupting renal afferent and efferent nerves thereby attenuating sympathetic nervous activity, is well-known as an innovative method for drug-resistant hypertension.⁹ The results of the Symplicity HTN-1 study (a nonrandomized proof-of-concept study) showed that RDN was a feasible, effective, and safe method for drug-resistant hypertension.¹⁰ Subsequently, the Symplicity HTN-2 study (a randomized clinical trial) showed BP in the RDN group reduced substantially more than that in medication-only group.¹¹ More recently, longer-term follow-up of the Symplicity HTN-1 and HTN-2 studies showed RDN offered safe and sustained reduction of BP to at least one or two years.^12,13 Other than its effect of marked and sustained BP reduction, many studies also found muscle sympathetic nervous activity, renal norepinephrine spillover, and plasma renin levels decreased in patients who underwent RDN.^14,15 With this in mind, one of the major underlying mechanisms of RDN on BP reduction may be its decreased effect on the SNS and RAS. However, data related to the details of the effects of RDN on RAS activity are not reported. Furthermore, many studies have indicated that angiotensin II is a potent stimulator of endothelin-1 (ET-1) production and exerts its hypertensive effects via interaction with the ET system.^16–19 Based on these considerations, we carried out a pilot study investigating the effect of RDN on RAS activity and ET-1, and explored the mechanism of BP reduction by RSD.

Methods

Animal preparation

All of experiments were approved by the animal experimentation ethics committee of Chongqing Medical University, following the guidelines of the National Institutes of Health for the care and use of laboratory animals. Standard food and water were given throughout the experimental period.

This study was performed on 10 healthy adult Chinese Kunming dogs of either sex, weight between 30–35 kg. The Chinese Kunming dogs are larger, more aggressive and fierce than beagles, spaniels and mongrels, with naturally high BP and sympathetic activity, and were the optimal experimental animals used for this study. Eligible Kunming dogs were older than three years and had a systolic BP of 140 mm Hg or more.

On the day of experiment, the Kunming dogs were generally anesthetized with 3% sodium pentobarbital of 30 mg/kg by intraperitoneal injection, followed by a maintenance dose of 5 mg/kg per hour. Penicillin was given intramuscularly before and after the ablation for the prevention of infection. The right femoral artery and vein were punctured under sterile conditions, then 8F and 6F sheaths were placed, respectively. An amount of 2000 IU unfractionated heparin was administered intravenously for anticoagulation. Blood sampling was obtained from right femoral vein and BP was monitored via the right femoral artery by connecting to the pressure transducer device before and 30 min after the ablation.

Renal angiogram and renal artery ablation

Bilateral renal angiography was performed using 6F JR4 Judkins catheter (Cordis Corporation Miami, Florida, USA). If renal artery abnormalities were found or diameters were below the minimum tolerable size (<4 mm, using the inner diameter of 6F JR4 Judkins catheter as reference), the Kunming dog was eliminated from the study. After confirmation of eligibility, bilateral RDN was performed with a saline-irrigated catheter (Biosense Webster, Diamond Bar, California, USA). The catheter was positioned into each renal artery via femoral access, and then 8–12 watts radiofrequency energy was applied both longitudinally and rotationally within the main stem of the renal arteries. Up to eight ablations were performed in each renal artery and ablative time for each point lasted up to 60–90 s. The temperature of tissue-electrode interface was maintained to 40°C by saline irrigated manually during the ablation procedure. After the ablation procedure, the angiography was redone to evaluate the morphology of renal artery, and also for the documentation of vessel patency and intact kidney perfusion.

Blood sampling assays

Before, 30 min, one month, and three months after ablation, respectively, 3 ml of blood sampling to measure plasma levels of renin, angiotensin II, and ET-1 was obtained in ethylenediaminetetraacetic acid (EDTA) from the right femoral vein. After high speed centrifugation, the blood samples were stored at –80°C until assay. The plasma levels of renin, angiotensin II, and ET-1 were assayed by radioimmunoassay.

Follow-up assessment

During the follow-up period, the condition of all Kunming dogs was observed daily. After the ablation procedure of one month and three months, follow-up assessments were conducted, which consisted of BP measurements via femoral artery, bilateral renal arterial angiography, and plasma levels of renin, angiotensin II, and ET-1 assay, as described above.

Statistical analysis

All data were expressed as mean±standard deviation (SD). Repeated measures analysis of variance (ANOVA) was used to compare BP, plasma levels of renin, angiotensin II, and ET-1 from baseline to 30 min, one month, and three months. A least significant difference (LSD)-t test was used for intergroup comparison. The relationship between plasma level of renin and angiotensin II, as well as plasma level of angiotensin II and ET-1 were assessed by Pearson correlation analysis. A two-sided p<0.05 was regarded as statistically significant. All of the statistical analyses were performed with SPSS statistical software (version17.0, Chicago, Illinois, USA).

Results

Ten Kunming dogs in preparation for RDN underwent renal angiography, but two Kunming dogs were excluded for anatomical reason (due to dual renal artery system). The remaining eight Kunming dogs underwent RDN and completed three months of follow-up.

Plasma renin, angiotensin II, and ET-1

The changes of plasma level of renin, angiotensin II, and ET-1 are presented in Figure 1. Repeated measures ANOVA testing showed that plasma levels of renin, angiotensin II, and ET-1 were significantly lower after the procedure than before the procedure (p=0.019 for renin, p=0.001 for angiotensin II, and p<0.001 for ET-1). Compared to those at baseline, RDN decreased plasma renin from 67.57±15.63 pg/ml to 61.88±13.30 pg/ml (p=0.049), 48.41±10.97 pg/ml (p=0.025), and 45.21±9.87 pg/ml (p=0.02), respectively. Consistent with the changes of renin, plasma angiotensin II fell from 54.07±11.75 pg/ml to 43.45±6.83 pg/ml, 38.22±6.24 pg/ml, and 38.88±6.62 pg/ml (p=0.004, 0.001 and 0.003, respectively). Additionally, plasma ET-1 decreased from an average of 28.8±6.75 pg/ml at baseline to 24.89±6.16 pg/ml, 17.98±7.04 pg/ml and 14.16±5.8 pg/ml at 30 min, one month, and three months of follow-up (p=0.007, p<0.001 and p<0.001, respectively).

Figure 1.

The plasma levels of renin, angiotensin II, and endothelin-1 (ET-1) in eight dogs before and after renal denervation (RDN), as shown in (a), (b), and (c), respectively.

The Kunming dogs underwent RDN exhibited lower plasma renin, angiotensin II and ET-1 levels at 30 min after procedure, were further reduced at one month, and persisted through subsequent assessments up to three months (Figure 2). Mean reductions in plasma renin were –5.7±6.8,–19.4±19.3, and –22.4±21 pg/ml, in plasma angiotensin II were –10.6±7.2, –15.9±8.8, and –15.2±9.6 pg/ml, and in plasma ET-1 were –3.9±3,–10.8±5, and –14.6±6.1 pg/ml, respectively. The progressive and sustained reduction of plasma level of renin was consistent with angiotensin II and ET-1 from baseline to three-month follow-up. Following Pearson correlation analysis, we found that plasma level of renin correlated closely with plasma level of angiotensin II (r=0.757, p<0.001, Figure 3). In addition, There was a significant linear relationship between plasma level of angiotensin II and ET-1 (r=0.712, p<0.001, Figure 3).

Figure 2.

Reductions of plasma renin-angiotensin system (RAS) and endothelin-1 (ET-1) at 30 min, one and three months after ablation. *p=0.049; §p=0.025; **p=0.02; †p=0.004; ¶p=0.001; ††p=0.003; ‡p=0.007; #p<0.001.

Figure 3.

Correlation of plasma level of renin and angiotensin II is shown in (a), and correlation of plasma level of angiotensin II and ET-1 is shown in (b) (r indicates the Pearson correlation coefficient).

BP

Systolic/diastolic BP was 160±19/98±15, 141±19/91±10, 130±13/86±13, and 124±9/82±6 mm Hg at baseline, 30 min, one month, and three months after the procedure, respectively. The repeated measures ANOVA test showed that both systolic and diastolic BPs were significantly lower after procedure than before procedure (p<0.001 for systolic and p=0.02 for diastolic BP). Following the LSD-t test, at all time points after procedure, both systolic and diastolic BPs were significantly lower than baseline BP, with the exception of the 30-minute diastolic BP (p=0.045, p<0.001 and p<0.002 for systolic BP; p=0.14, p=0.036 and p₌0.014 for diastolic BP, respectively; Figure 4). RDN reduced BP at 30 min, one month and three months by –19±22/–8±13, –30±13/–13±14, and –36±20/–16±14 mm Hg, respectively.

Figure 4.

Systolic blood pressure (SBP) and diastolic blood pressure (DBP) at baseline, 30 min, one and three months of ablative procedure (*p<0.05 after versus before renal denervation (RDN)).

Renal angiogram

There was no evidence of renal artery dissection, stenosis or other abnormalities occurring in the Kunming dogs which underwent RDN, either during the procedure and follow-up period. Renal angiographic studies identified focal artery irregularities immediately after radiofrequency energy delivery, none of which were judged as flow limiting at procedure termination (Figure 5(b)). One-month follow-up angiograms showed that almost all ablated lesions had disappeared and the renal artery wall had recovered smoothly (Figure 5(c)).

Figure 5.

The results of angiography before, immediately, and one month after renal denervation (RDN), as shown in (a), (b), (c), respectively.

Discussion

The main findings of the current study were as follows: (a) RDN caused a substantial and uniform reduction in plasma levels of renin, angiotensin II, and ET-1; (b) there was a significant linear relationship between plasma level of renin and angiotensin II, as well as plasma level of angiotensin II and ET-1; (c) RDN utilizing saline-irrigated catheter caused a progressive and substantial BP reduction without ablation-related complications; (d) repeat angiograms showed that focal artery irregularities immediately after radiofrequency energy delivery all disappeared after one month.

Effects of RDN on RAS activity and ET-1

Previous research studies proved that SNS and RAS activation are crucial and interact closely in the pathogenesis of cardiorenal diseases. They showed that renal sympathetic nerves terminate on various elements in the kidney, including the vessels, renal tubules, and juxtaglomerular granular cells.²⁰ Based on this consideration, many cardiorenal diseases changing renal sympathetic nerve activity (RSNA) could directly influence the functions of innervated renal effector units. Increases in RSNA raise plasma RAS levels by stimulating renin release from β1-adrenoreceptors on juxtaglomerular granular cells, decrease urinary sodium and water excretion by increasing renal tubular water and sodium reabsorption through the nephron, and decrease glomerular filtration rate by constricting the renal vasculature: in turn this contributes to the development of cardiorenal diseases.^21,22

Many β-adrenoreceptors blockers can reduce plasma renin activity in patients and experimental animals,^23,24 and are found to be more effective in patients with higher renin profiles.²⁵ Blocking the effect of renal sympathetic nerves has been proved to be an effective way to affect RAS, accordingly, blocking the renal sympathetic nerve pathway should present the same effect. Kassab et al. described how RDN prevented the transient increase of plasma renin activity in the obesity-induced hypertension dog model.²⁶ Moreover, variables of the circulating RAS are undetectable after bilateral nephrectomy.²⁷ A recent human postmortem histologic study demonstrated that the renal sympathetic nerves are distributed around the renal artery and a great proportion of renal sympathetic nerves had close proximity to the lumen-intima interface which should thus be accessible via renal artery interventional approaches such as catheter ablation.²⁸ Therefore, if endovascular RDN ablates the renal sympathetic nerves in the adventitia successfully, theoretically, the plasma levels of RAS will be decreased after the procedure. As described in the results, RDN indeed caused a uniform decrease in plasma renin and angiotensin II. Furthermore, changes of plasma renin and BP in the present study were similar to those in the study of Schlaich et al.¹⁴ which showed that BP reduction was accompanied by halving of renin activity after RDN in patients with refractory hypertension.

Renin is recognized as an activator of RAS and angiotensin II is the most powerful biologically active product of the RAS. Angiotensin II directly constricts vascular smooth muscle cells, enhances myocardial contractility, stimulates aldosterone production, stimulates release of catecholamines from the adrenal medulla and sympathetic nerve endings, increases sympathetic nervous system activity, and stimulates thirst and salt appetite.²⁹ Also, many studies mentioned in the introduction have indicated that angiotensin II is a potent stimulator of ET-1 production and exerts its hypertensive effects via interaction with the ET system. In our study, RDN caused a substantial and uniform reduction in plasma levels of renin, angiotensin II, and ET-1, accompanying a BP decrease. Additionally, there were significant linear relationship between plasma level of renin and angiotensin II, as well as plasma level of angiotensin II and ET-1. These findings suggested that RDN attenuating the renal sympathetic activity could decrease the plasma level of renin, which may affect the production of angiotensin II and this later could then change the plasma levels of ET-1. Decreased activity of the SNS is one of the major underlying mechanisms of RDN on the marked and sustained BP reduction, but the precise mechanisms that cause the fall in BP in the short-term and, in particular, long-term remain elusive. Therefore, our study offered a new angle to explore the therapeutic mechanism of RDN. Furthermore, excessive sympathetic drive and inappropriate renin stimulation are hallmarks not only for refractory hypertension, but also for chronic heart failure, progressive kidney disease, sympathetically mediated insulin resistance: all diseases where sympathetic nervous activity is considered to play a key role in their development and progression. If the observations in this study continue to hold true in future clinical studies, RDN might be extended in the future to the treatment of various cardiovascular diseases.

Renal angiogram

As we know, a saline-irrigated catheter which has been used to ablate cardiac arrhythmia has the advantage of being less likely to cause thrombus or char formation at the ablation site. And recently RDN utilizing the saline-irrigated radiofrequency ablation catheter has been demonstrated to safely reduce BP in patients with refractory hypertension by Ahmed et al.³⁰ Compatible with this clinical report, in the present study, RDN by saline-irrigated radiofrequency ablation catheter decreased BP without ablation-related complications occurring. Although angiography results showed there were slight vessel irregularities caused by radiofrequency energy delivery, the lesions disappeared at first month of follow-up. Our angiography results were similar to those of the study by Steigerwald et al.³¹ which conducted RDN using the Symplicity Catheter System. In Simplicity trials, renal artery aneurysm or stenosis was seldom seen as judged by computed tomography (CT) or magnetic resonance imaging (MRI) angiography. Of note, the basis of catheter-based RDN is an intended tissue injury to renal sympathetic nerve fibers caused by heat generated by radiofrequency energy. Nevertheless, unintended tissue injury to surrounding vascular structure causing edema, inflammation and fibrosis is conceivable, as are focal artery irregularities shown in our repeat angiograms immediately after radiofrequency energy delivery. Although the incidence of ablation-related complications seems to be low, unintended tissue injury to surrounding vascular structures may increase potential risks. Further, device technology minimizing either mechanical or heat related vascular tissue injury needs to be explored.

Study imitations

Some limitations should be considered in interpreting the present results. Firstly, norepinephrine spillover measurement may help estimating the activity of the efferent renal sympathetic nerve, but our experimental facility did not allow using radioisotopes. Secondly, due to sample size of this study being relatively small, follow-up duration was relatively short, and differences between the Kunming dogs and hypertensive human subjects exist. Whether RDN could still significantly decrease the plasma levels of RAS and ET-1 in patients deserves further clinical studies, which will be clarified in the on-going SouthWestern China renal Artery sympathetic Nerve ablation study of Hypertension (SWAN-HT) study (NCT01417221) with 70 resistant hypertensive patients. Furthermore, a previous study³² indicated that local RAS contents were inhomogeneous and tissue RAS exerted particularly more important roles than circulating RAS in cardiovascular pathophysiological conditions. However, we investigated the effects of RDN on the RAS via determining the changes in circulating levels of renin, angiotensin II and ET-1, therefore, further studies about effects of RDN on tissue RAS are necessary. Finally, although angiograms showed focal artery irregularities immediately after radiofrequency energy delivery all disappeared after one month, some local tissue damages were not apparently identified by angiograph. Therefore, it would be a bit premature to conclude that RDN using saline-irrigated catheter would not cause any ablation-related complications and further histological examinations are significant.

Conclusion

In conclusion, RDN caused a substantial reduction of BP, and significantly decreased the plasma levels of the RAS and ET-1, which may be implicated in the mechanisms of marked and sustained BP reductions by RDN.

Footnotes

Conflicts of interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported in part by research grants from the Science and Technology Committee of Yuzhong district, Chongqing, China (grant number: 20110301); and the Foundation for Key Research of Chongqing Municipal Health Bureau, China (grant number: 2011-1-045).

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