Preclinical Evaluation of the Renal Nerve Anatomy Renal

Preclinical Evaluation of the Renal Nerve: Anatomy, Renal Nerve Distribution and Animal Models Aloke Finn, M. D. CVPath Institute Inc. Gaithersburg, MD, USA

Disclosure Statement of Financial Interest Within the past 12 months, I or my spouse/partner have had a financial interest/arrangement or affiliation with the organization(s) listed below. Employment in industry: No Honorarium: Abbott Vascular, Boston Scientific, Lutonix, Terumo Corporation, and W. L. Gore. Institutional grant/research support: 480 Biomedical, Abbott Vascular, Atrium, Bio. Sensors International, Biotronik, Boston Scientific, Cordis J&J, GSK, Kona, Medtronic, Micro. Port Medical, Celo. Nova, Orbus. Neich Medical, Re. Core, SINO Medical Technology, Terumo Corporation, and W. L. Gore. Owner of a healthcare company: No Stockholder of a healthcare company: No

Cumulative Distance From Nerves to Lumen 100 Cumulative percentile of nerves 90 80 70 90 th percentile Proximal 6. 77 mm / Middle 6. 64 mm /Distal 5. 27 mm 75 th percentile Proximal 4. 67 mm / Middle 4. 49 mm / Distal 3. 24 mm 75% of sympathetic nerves located a mean of 4. 28 mm from lumen 60 50 50 th percentile Proximal 2. 84 mm / Middle 2. 58 mm / Distal 1. 81 mm 40 30 Proximal (n=3070) 20 Middle (n=2952) Distal (n=2008) 10 0 ≤ 0. 5 ≤ 1. 0 ≤ 1. 5 ≤ 2. 0 ≤ 2. 5 ≤ 3. 0 ≤ 3. 5 ≤ 4. 0 ≤ 4. 5 ≤ 5. 0 ≤ 5. 5 ≤ 6. 0 ≤ 6. 5 ≤ 7. 0 ≤ 7. 5 ≤ 8. 0 ≤ 8. 5 ≤ 9. 0 ≤ 9. 5 ≤ 10. 0 Distance from arterial lumen (mm) Sakakura K, et al. J Am Coll Cardiol 2014

0 -<1 1 -<2 2 -<3 3 -<4 4 -<5 5 -<6 6 -<7 7 -<8 8 -<9 9 -<10 ≥ 10 Total No. of nerves No. of arterial section Renal Artery Diameter (mm) Proximal Middle Distal Post Bifurcation Total 218 831 549 469 314 249 163 120 91 58 8 3070 82 244 892 553 372 284 203 169 86 71 71 7 2952 76 227 877 324 213 133 115 39 36 29 14 1 2008 62 801 1023 261 97 54 29 15 4 7 7 1 2299 80 1490 3623 1687 1151 785 596 386 246 198 150 17 10329 300 4. 4± 0. 9 4. 0± 0. 7 4. 1± 0. 9 3. 6± 1. 4 Mean number of nerves /1 artery of 1 section 60 50 40 30 20 10 0 63% Sakakura K, et al. , JACC 201464: 634 -43 Distance from lumen to nerve (mm) Mean Distance from lumen to nerves (mm) P<0. 05 5 p<0. 05 Vs proximal and middle Vs post bifurcaion 4 3 P<0. 05 vs proximal and middle and distal 2 1 Proximal 39. 6 Middle 39. 9 Distal 33. 6 Post bifurcation 14. 7/artery 14. 7 0 Proximal 3. 4 Middle 3. 1 Distal 2. 6 Post bifurcation 1. 54

Proposed Diagram of Renal Artery and Circumferential Peri-Arterial Nerve Location Sakakura K, et al. , JACC 201464: 634 -43

Distribution and Density of Renal Sympathetic Nerves Distribution of nerves stratified according to total number (each green dot represents 10 nerves), relative number as percent per segment, and distance from the lumen in relative (A) proximal, (B) middle, and (C) distal location. Figure prepared using raw data from Sakakura et al. (4), and from raw data provided by M. Joner

Distribution of Nerve Aorticorenal ganglion Renal artery (accessary) Renal artery Renal plexus overlying renal arteries Aortic plexus Lusch A et al. , J Urol. 2014 Apr; 191(4): 1060 -5.

Variation in Anatomy Among Human and Pig Renal Arteries Human renal artery – anatomical variants Ø About 70% of the population have a single renal artery 1 Ø Accessory renal arteries are the most common variant present in about 1/3 of the population 2 Ø Prehilar (early) branching is a normal variant and present in about 10 -20% of the population (32% on right RA, 25% on left RA and 22% on both sides)3, 4, 5 Ø Pig renal arteries more often have early branches 1 Hazirolan et al. , Diagn Interv Radiol, 2011 et al. , Diagn Angiography, 1986 3Özkan et al. , Diagn Interv Radiol, 2006 4 Chai et al. , Korean J Radiol, 2008 5 Patil et al. , Nephrol Dial Transplant, 2001 2 Kadir

Distribution of Nerves in Accessory Renal Arteries Aorta Accessory Renal Artery Mean. Number. Of Of. Nerves. Per. Quadrant Mean 18. 0 16. 0 14. 0 12. 0 10. 0 8. 0 6. 0 4. 0 2. 0 0. 0 main renal artery post bifurcation accessory arteries Ventral 11. 00 44 3. 5 Dorsal 6. 20 3. 6 1. 1 Korean J Radiol. 2010; 11(3): 346– 354 Superior 10. 80 3. 33. 3 Inferior 9. 40 3. 8 2. 5 Sakakura K, et al. , JACC 201464: 634 -43

Assessment of Renal Nerves in a Healthy Swine Model Proximal Middle Distal Postbifurcation Post 1 Post 2 Diameter of nerves (shortest) Aorta Renal artery Kidney 1 st bifurcation 2 nd bifurcation Distance from lumen of renal artery to nerve Ureter

The Percentage of Nerves Stratified by Distance from Lumen of Renal Artery Proximal Distal 6 mm 4 mm 2 mm Mean number of nerves per artery Any size ≤ 2 mm 2 -4 mm 4 -6 mm >6 mm Proximal 22 14 10 11 Middle 23 11 7 4 ≤ 2 mm 2 -4 mm 100 88% 80 52% 60 4 -6 mm 88% 86% 40 20 20 Mid 99% 96% 58% 60 38% Prox Intermediate Nerves (≥ 50 µm) 80 62% Post 2 22 4 4 2 >6 mm 100 40 0 Post 1 23 3 3 2 (%) Any nerves (%) Distal 30 10 5 5 Dist Post 1 Post 2 0 32% Prox Mid Dist Post 1 Post 2

Mean number of nerves per artery (nerves divided by number of arteries in post bifurcation sections) Mean number of nerves (≥ 50 µm) within 2 mm per artery 14 P = 0. 001 12 10 8 6 4 2 0 4. 3 5. 5 6. 8 4. 0 2. 5 Prox Mid Dist Post 1 Post 2 The largest proportion of intermediate size nerves are found in the distal main renal artery close to the bifurcation

Anatomy of Renal Sympathetic Nerves in a Healthy Swine Model Ganglion Ø Nerve size is greater in proximal than distal and post-bifurcation Target for ablation Aorta Renal artery Kidney 1 st bifurcation 2 nd bifurcation Ureter Ø Nerves are located close to the renal artery in distal and postbifurcation compared to proximal segments. Ø Nerves also branch at the site of bifurcation.

Summary • Maximum number of Nerves are located close to the distal renal artery. • The size of nerves diminishes from proximal to distal and postbifurcation renal artery segments • intermediate size nerves (≥ 50μm) are most frequently located within 2 mm of the renal artery lumen in the distal and postbifurcation segments • The maximum number of intermediate sized (>50μm) nerves within 2 mm are in the distal main renal artery • Therefore, renal denervation should be performed in the distal main renal artery, with or without the middle or proximal renal arteries.

Acknowledgments Funding CVPath Institute Inc. CVPath Institute Kazuyuki Yahagi, MD Hiroyoshi Mori, MD Sho Torii, MD, Ph. D Emanuel Harari, MD Maria Romero, MD Robert Kutz, MS Russ Jones Abebe Atiso, HT Jinky Beyer Lila Adams, HT Frank D Kolodgie, Ph. D Aloke V. Finn, MD Washington DC