From July 2011 to December 2013, we performed a prospective observational study using 123I–mIBG scintigraphy as a parameter of renal sympathetic activity in patients with RHT undergoing RDN. Objectives were to compare measures of renal 123I–mIBG uptake (uptake at 15 min and washout between 15 min and 4 h) on planar and single photon emission computed tomography-CT (SPECT-CT) images, changes in office based BP and ambulatory BP measurements (ABPM) and neurohormonal activation before and 6 weeks after RDN.
Patients
In the present study, we enrolled 21 consecutive patients aged 40–70 years with a clinical indication for RDN because of therapy resistant hypertension defined as a mean daytime BP ≥ 150/100 mmHg despite the use of three or more anti-hypertensive drugs including or with intolerance to a diuretic [8]. Secondary causes of hypertension (e.g., renal artery stenosis, pheochromocytoma, primary aldosteronism, and hyper- or hypothyroidism) and abnormal renal artery anatomy, including the presence of accessory renal arteries, were ruled out prior to the intervention. Patients with renal insufficiency (estimated glomerular filtration rate (eGFR) < 45 mL/min/1.73 m2) or proteinuria (> 1 g/24 h) or having a pacemaker, implantable cardioverter-defibrillator (ICD), atrial fibrillation, or type 1 diabetes mellitus were excluded.
Antihypertensive treatment was performed according to international guidelines and included instructions on dietary sodium restriction, physical activity, and instructions to remain compliant to antihypertensive medication [8, 9]. Six weeks prior to the first measurements, patients were screened to assess eligibility for study participation. Patients were deemed eligible for study participation if they were at least 3 weeks on stable BP lowering medication prior to the first study visit. BP lowering medication was kept unchanged throughout the study until the final visit 6 weeks after RDN.
When fully informed and willing to participate, patients were asked to provide written informed consent. Six weeks hereafter, office BP and ABPM were measured. Patients were required to maintain the same antihypertensive drug regimen throughout study participation. This study was a part of a larger effort to assess the sympaticolytic potential of RDN with the predetermined idea to assess the effects of RDN on renal 123I–mIBG uptake and washout.
For reference, we used data of five patients (aged 39–66 years) in whom 123I–mIBG was performed of the kidney allograft after recent kidney transplantation (0.1 to 1.5 years after transplantation), whose detailed characteristics are described elsewhere [7]. In summary, all these surgically denervated kidneys functioned well with creatinine clearance rates (calculated from 24 h urine collections) ranging from 54 to 128 ml/min. As a negative control, we also included 123I–mIBG data from a patient with complete renal denervation after autologous kidney transplantation for renal artery stenosis [10]. Although 123I–mIBG is primarily cleared via the kidneys, we have shown that both the cardiac as well as the renal 123I–mIBG parameters (i.e., late heart-to-mediastinal ratio, renal uptake, and renal washout) are not influenced by kidney function [7, 11].
Study protocol
The study protocol met the ethical guidelines of the Declaration of Helsinki (originally adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964 and last amended in Fortaleza, Brazil 2013) and was approved by the local ethics committee of the Academic Medical Center at the University of Amsterdam (number NL.36755.018.11). All patients gave oral and written informed consent before study inclusion.
Renal sympathetic denervation procedure
The renal denervation procedure was performed via the femoral artery approach by a single highly experienced interventional radiologist (JAR) with > 5 RDN procedures before this study was initiated. RDN was performed by use of radiofrequency energy delivered by the Symplicity renal-denervation catheter (Medtronic Inc., Santa Rosa, California, USA). Prior to the procedure, midazolam 1.0 mg and metoclopramide 10 mg was given intravenously. After inserting a 6 F introducer sheath in the right femoral artery, the guiding catheter was introduced in the aorta and an aortagram was made. The guiding catheter was advanced in the right and left renal artery in no pre-specified order. The denervation catheter was introduced in the renal artery via the guiding catheter. After nitroglycerine 0.2 mg and fentanyl 0.02 mg intravenously, catheter ablations were performed in a helical pattern with the goal of at least 4–6 ablations per renal artery to cover each short axis transaxial quadrant, according to the user’s instruction of the device. No peri-procedural complications occurred.
Blood pressure monitoring
At baseline and 6 weeks after RDN 24 h ABPM was performed using the Spacelabs 90,217 ABPM monitoring device (Spacelabs Healthcare, Issaquah, Washington, USA). During day time between 06.00 am and 23.00 pm, measurements were performed every 15 min and at night-time (i.e., 23.00 pm and 6.00 am) every 30 min. BP readings were accepted when the success rate of the measurements was minimally 70% per 24 h. Patients were blinded to their BP readings. Instructions were given to continue usual daily activities during 24 h of BP recording, but avoiding strenuous exercise. Office brachial BP using appropriate cuff-sizes was measured with a validated semi-automated oscillometric device (Omron 705it, Omron Healthcare Europe BV, Hoofddorp, The Netherlands), while seated and after 5 min rest in a quiet room, three times at 1 min intervals by a trained research assistant or physician. The mean of the last two measurements was recorded as representative of office brachial BP. No BP measurements were performed in the kidney transplant recipient group.
Blood and urine analysis
Plasma renin activity (PRA) (μgA1/L/h) was analyzed using radioimmunoassays. Urine and plasma epinephrine, norepinephrine (NE), metanephrine, and normetanephrine were analyzed using liquid chromatography-mass spectrometry. Epinephrine and NE and were obtained in supine as well as after 5 min in standing position. The delta of supine minus standing position was calculated. Urinary sodium excretion (mmol/24 h), urine creatinine (μmol/L), was calculated from 24 h urine collections obtained before and 6 weeks post-RDN.
123I–mIBG scintigraphy
The protocol of the renal 123I–mIBG scintigraphy has been previously described [7]. In summary, 2 h prior to the administration of 185 MBq (5 mCi ± 10%) 123I–mIBG (AdreView™, GE Healthcare, Eindhoven, the Netherlands) patients received 100 mg potassium-iodide to block thyroid uptake of “free” 123I. In addition subjects were given a single oral dose of furosemide retard 60 mg to promote the urinary excretion of 123I–mIBG. No specific instructions on fluid intake were given to enhance excretion of 123I–mIBG. Anterior and posterior planar semi-whole body images were performed at 15 min and 4 h after administration of 123I–mIBG. A vial with a reference amount of radioactivity of 123I was included in the planar images. Additionally, at 4 h post-injection (p.i.), SPECT-CT (low dose) was performed. The CT-images were used for an adequate anatomical registration of 123I–mIBG uptake.
Since we recently showed that uptake at 15 min p.i. of 123I–mIBG and washout between 15 min and 4 h can detect renal sympathetic reinnervation over time after transplantation, we report in this study the 123I–mIBG uptake on the 15 min p.i. images and analyzed the mean counts/pixel for calculation of washout between 15 min and 4 h [7].
123I–mIBG imaging procedures
The planar images were acquired with a 20% energy window centered at 159 keV, using medium-energy collimators. Anterior and posterior planar semi-whole body acquisitions were used to create geometrical mean images.
123I–mIBG image analysis
An investigator (LCD) analyzed the geometric mean (GM) planar images (Hybrid Viewer™, Hermes Medical Solutions, Stockholm, Sweden) by manually drawing regions of interest (ROI) for kidneys, muscle (M. quadriceps femoris), and the 123I vial. A predefined and fixed ROI for the muscle (50 pixels) was used for all patients.
We analyzed the counts of the left kidney only since scatter or overlay of the liver with a high uptake of 123I–mIBG resulted in poor delineation of the right kidney. Mean counts per pixel per ROI were used to calculate 123I–mIBG uptake: the relative uptake between kidney (specific) versus muscle (nonspecific) quantifies neural uptake of 123I–mIBG and reflects neuron function that results from 123I–mIBG uptake, storage and release. These can be derived using mean counts from the 15 min and 4 h p.i. GM images and the 4 h p.i. 123I–mIBG SPECT-CT images. Washout (WO) between 15 min and 4 h p.i. based on GM images reflects sympathetic activity and was calculated from the kidney-to-muscle ratio between 15 min and 4 h p.i.. Formulas to calculate uptake and washout were
$$ \mathrm{Relative}\ \mathrm{uptake}=\frac{\mathrm{kidney}\ \left(\mathrm{specific}\right)-\mathrm{muscle}\ \left(\mathrm{non}-\mathrm{specific}\right)}{\mathrm{muscle}\ \left(\mathrm{non}-\mathrm{specific}\right)} $$
$$ \mathrm{Washout}=\frac{\left(\ \frac{\mathrm{uptake}\ \mathrm{kidney}\ 15\ \min }{\mathrm{uptake}\ \mathrm{muscle}\ 15\min}\right)-\left(\frac{\mathrm{uptake}\ \mathrm{kidney}\ 4\ \mathrm{h}\ }{\mathrm{uptake}\ \mathrm{muscle}\ 4\ \mathrm{h}}\right)\ }{\left(\ \frac{\mathrm{uptake}\ \mathrm{kidney}\ 15\ \min }{\mathrm{uptake}\ \mathrm{muscle}\ 15\min}\right)}\mathrm{x}100\% $$
The percentage uptake of the injected dosage of 123I–mIBG was calculated using the actual injected dose and mean counts per pixel in relation to the activity in the 123I–vial. Washout [WO) in the left kidney was calculated from 15 min and 4 h images using skeletal muscle as a reference.
A secondary analysis was focused on the SPECT-CT images. In this method, the transverse CT images were used to optimize anatomical delineation of the kidney contours. The main advantage of this method is the availability of anatomical information obtained from the low dose CT, allowing for a superior delineation of kidneys and subsequently a potential better estimation of the renal 123I–mIBG uptake. ROIs were drawn on the CT-images along the contours of kidney cortices, excluding the calyces. ROIs were then fused into volumes of interest (VOIs) and copied to the co-registered SPECT. Mean counts/voxel expressed 123I–mIBG uptake. VOIs in muscle served as background activity.
Based on the difference in 123I–mIBG uptake, we divided patients with a positive change in 123I–mIBG uptake, i.e., indicating an increase in 123I–mIBG uptake or washout and those with a negative change, i.e., a decrease in 123I–mIBG uptake or washout after RDN.
Statistical analysis
This study was part of a larger effort to study sympatholytic effects of RDN. The sample size has been described elsewhere [12]. Data are presented as medians and interquartile ranges (IQR with 25 and 75 percentiles) and comparisons were performed by non-parametrical tests (Wilcoxon signed rank tests as well as the Mann–Whitney U test). P values below 0.05 were considered statistically significant. All analyses were performed using IBM SPSS Statistics software for Windows version 21.0 (IBM Corp. Armonk, New York, USA).