The study methodology and the primary BP outcomes have been published elsewhere . In this predefined sub-study, a total of nine Ossabaw swine were randomly allocated to catheter-based RF RDN receiving either bilaterally four (RF-4), eight (RF-8), or twelve (RF-12) RF-ablation lesions (Fig. 1). The second group of three Ossabaw swine underwent combined surgical and chemical RDN. At baseline (5 months before RDN), a telemetry device was surgically implanted during anesthesia in all 12 Ossabaw swine to monitor and acquire hemodynamic data continuously throughout the study period. Further, at baseline and before termination (3 months after RDN), the treatment group animals underwent an HC with blood sampling immediately before (pre-HC) and after HC (post-HC). Urine samples were drawn after the HC. At termination, all swine were sacrificed to measure renal norepinephrine concentrations and for histopathological examinations. Ten domestic swine served as controls for comparison of kidney norepinephrine (NEPI) concentrations of treated and untreated animals. The study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals under approved institutional animal care and use committee-approved protocol.
All animals fed the same high-calorie diet causing an increase in bodyweight (+ 51.8 ± 13.3 kg, p < 0.001) (Fig. 2), cholesterol (+ 2.547 ± 1.692 mmol/l, p = 0.002), and blood glucose levels (+ 1.248 ± 1.448 mmol/l, p = 0.032) throughout the study . Further, hypertension was developed in all pigs (systolic/diastolic BP at baseline 142.8 ± 5.2/107.6 ± 5.2 versus 169.5 ± 6.9/128.3 ± 5.7 mmHg at RDN, p < 0.001). Renal function remained unchanged during the study (creatinine at baseline 0.027 ± 0.01 mmol/l versus 0.026 ± 0.011 mmol/l at termination, p = 0.5).
Telemetric hemodynamic monitoring
In all animals, implantable telemetry devices (PhysioTel Digital model L11; Data Sciences Int.[DSI], St Paul, MN, USA) were used to monitor and collect hemodynamic parameters. The transmitters were implanted in an intermuscular pocket in the neck area using standard surgical techniques and covered with surgical mesh to facilitate tissue healing. The systemic BP catheter was tunneled and inserted into the carotid artery close to the carotid bifurcation. The negative ECG biopotential lead was introduced into the jugular vein and advanced caudally to place the distal tip at the junction of the superior vena cava and right atrium. The distal end of the positive ECG lead was tunneled subcutaneously and secured at the left lateral costal arch level to acquire an ECG in lead II configuration. For the analyses of hemodynamic data, we calculated the 24-h, daytime (0600–1800), and nighttime (1800–0600) average BP and heart rate values. Furthermore, the minimum and the maximum values during the 24-h recording period were documented.
During anesthesia, an HC was induced by a 5-min intravenous infusion of sodium nitroprusside (2 μg/kg of bodyweight per min, diluted in glucose 5%), which reduces pre- and afterload as it releases nitric oxide, causing arterial and venous vasodilatation. The HC was performed 5 months before RDN during telemetry implantation and 3 months after RDN prior to termination.
Nine animals underwent minimally invasive catheter-based RDN using a multielectrode RF catheter (EnligHTN Renal Artery ablation Catheter, Abbott, MN, USA), whereas three animals were assigned to combined surgical and chemical RDN. For catheter-based RDN, an 8 Fr sheath was placed in the femoral artery under sterile conditions. Selective angiography and quantitative vascular analysis were performed on both sides to assess anatomical eligibility for RDN. Appropriate renal artery anatomy included main renal arteries between 4 and 8 mm in diameter and with sufficient length in the absence of accessory renal arteries. The RDN catheter was introduced into the main renal artery via a guide catheter and was connected with the dedicated generator (model 100,078,171). The animals bilaterally received four (n = 3), eight (n = 3), or twelve (n = 3) ablations. Before and 5 min post-treatment, nitroglycerin was injected into the renal artery. A post-treatment angiography was performed before removing the sheath. Three animals underwent combined surgical and chemical RDN requiring median laparotomy. First, all visible nerves surrounding the renal arteries were disrupted. Then, the top layers of the renal artery adventitia (2–4 mm) were stripped, and finally, the surface was covered with a phenol/ethanol solution (10–20%) for 10–15 min causing neurolysis.
Blood and urine analysis
Blood samples from the renal artery and vein were drawn immediately before and after the 5-minute intravenous infusion of sodium nitroprusside. Urinary catheterization was used to collect urine samples after the HC. Renin concentrations were measured using a porcine renin ELISA kit (NeoBiolab, Cambridge, MA, USA). The estimated renal renin AV-Δ was calculated by subtracting the arterial from venous renin concentration.
At the end of the 8-month study period, a necropsy was performed in every animal by a Diplomat, American College of Veterinary Pathologists, as described previously in detail .
Kidney tissue norepinephrine concentration
The left and right kidney were removed, and after examination, the renal tissue was immediately placed into liquid nitrogen at − 20 °C and homogenized. A sample of the homogenate was thawed and extracted with ammonium acetate buffer. The extracts were purified by a multisolvent solid-phase extraction (SPE) purification process. For analysis, the eluate from the SPE cartridges was injected into high-performance liquid chromatography (HLPC) instrument with electrochemical detection (ECD) system.
Five renal arteries were selected for the evaluation of nerve distribution. The renal arteries were cut into equal transverse segments (3–4 mm) before being fixed and embedded in paraffin. All sections were processed using alcohols and xylenes, cut on a rotary microtome at 5 µm, and then stained with hematoxylin and eosin, modified Movat Pentachrome, and neurofilament protein (NFP). All slides were imaged with the Axio Scan.Z1 slide scanner equipped with Zeiss Efficient Navigation (ZEN) 2012 software (Zeiss, Jena, Germany). Measurements were performed with HALO software (Indica Labs, Corrales, MN, USA).
Data are presented as mean ± standard deviation (SD) and numbers (%) unless otherwise specified. A two-tailed p value < 0.05 was considered to be statistically significant. The Mann–Whitney U, Wilcoxon, and Kruskal–Wallis test by ranks were used for non-normal data. Pearson’s correlation coefficient was calculated for baseline renal renin AV-Δ and change in systolic, mean, and diastolic BP. STATA, version 16.1 (StataCorp LLC, College Station, TX, USA) and Minitab, Version 17.0.1 (Minitab Inc., State College PA, USA) were used for statistical analysis. Graphs were created using GraphPad Prism, version 8.0.1 (GraphPad Software, San Diego, CA, USA).