Supplemental methods
Immunohistochemistry and Immunofluorescence
To obtain accurate nerve contour for nerve size measurements, staining for neurofilament protein
(NFP) (dilution 1: 40; Dako, Carpinteria, CA) was applied in selected cases. The nerve’s shortest
diameter was used for nerve size delineation. Also, the correlation between nerve counts
identified by H&E stain and those identified by NFP stain was examined to validate the
assessment of nerves by H&E stain.
Paraffin-embedded slides were de-paraffinized and hydrated. For antigen retrieval, sections
were boiled under pressure in EDTA for 15 minutes. After rinsing, the sections were incubated
with a mixture of mouse anti-tyrosine hydroxylase (TH) (1:100; EMD Millipore, Billerica, MA)
and rabbit anti-calcitonin gene-related peptide (CGRP) (1: 2000; Sigma-Aldrich, St. Louis, MO)
overnight at 4 °C, and then incubated with a mixture of anti-mouse Alexa Fluor 555 (1:150; Life
technologies, Carlsbad, CA) and anti-rabbit Alexa Fluor 488 (1:150; Life technologies, Carlsbad,
CA) for 60 minutes at 25 °C.
DAPI was used as nuclear counter stain (1:1000; Life
technologies, Carlsbad, CA) for 10 minutes at 25 °C. TH, which converts tyrosine to DOPA, was
used for the recognition of efferent nerves, whereas CGRP, which serve as neurotransmitters in
sensory nerves, was used for the recognition of afferent nerves (1-3).
Confocal microscope images of dual-stained TH positive and CGRP positive areas were
acquired randomly from four different sets of nerve fascicles per section at 40 x magnification
using a LSM-700 microscope (Zeiss, Oberkochen, Germany). Each image was processed and
analyzed with Zen imaging software (2011 edition, Zeiss). Semi-quantification of positive
staining in each examined channel was performed using a histogram function with background
correction and data expressed as ratio of TH/CGRP positive area.
Furthermore, to confirm the existence of sympathetic nerve fibers in intra-renal tissue, dual
immunofluorescence staining using antibodies targeted at anti-TH and anti-CGRP was also
performed in selected sections.
References
1.
Tokushige N, Markham R, Russell P, Fraser IS. Different types of small nerve fibers in
eutopic endometrium and myometrium in women with endometriosis. Fertil Steril
2007;88:795-803.
2.
Burgi K, Cavalleri MT, Alves AS, Britto LR, Antunes VR, Michelini LC. Tyrosine
hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous
evaluation in different tissues of hypertensive rats. American journal of physiology
Regulatory, integrative and comparative physiology 2011;300:R264-71.
3.
Liu L, Barajas L. The rat renal nerves during development. Anat Embryol (Berl)
1993;188:345-61.
Supplemental Tables
Supplemental Table 1. Distribution of nerves stratified by total number and distance from
lumen based on anatomical location in Post bifurcation segments.
Whole distribution (n=2299 nerves)
Distance from lumen to
nerve (mm)
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
Ventral
Dorsal
Superior
Inferior
Total
265
263
52
31
18
4
0
0
0
0
0
633
255
240
45
8
6
1
0
0
0
0
0
555
128
247
75
32
11
10
6
0
2
5
0
516
153
273
89
26
19
14
9
4
5
2
1
595
801 (34.8)
1023 (44.5)
261 (11.4)
97 (4.2)
54 (2.3)
29 (1.3)
15 (0.7)
4 (0.2)
7 (0.3)
7 (0.3)
1 (0.04)
2299
Comparison between Ventral, Dorsal, Superior, and Inferior based on each renal artery (n=40 renal arteries)
P value
P value for
for
Ventral
Dorsal
Superior
Inferior
P value for
All (n=40)
Ventral vs. Superior
(n=40)
(n=40)
(n=40)
(n=40)
overall
Dorsal
vs.
Inferior
Mean
Number of
nerves per
branch (n)
Mean
Distance
from lumen
to nerve
(mm)
14.7±6.0
4.0±2.2
3.6±2.5
3.3±2.2
3.8±2.0
0.12
-
-
1.54±0.45
1.34±0.57
1.19±0.32
1.66±0.74
1.74±0.86
<0.001
0.50
1.00
Friedman test was used.
Supplemental Table 2. Comparison between Ventral, Dorsal, Superior, and Inferior based
on each accessory artery (n=6 accessory arteries)
Number of nerves per
accessory artery (n/section)
Mean distance from
accessory artery lumen to
nerve (mm)
Friedman test
Overall
(n=6)
Ventral
(n=6)
Dorsal
(n=6)
Superior
(n=6)
Inferior
(n=6)
P value
10.4±5.9
3.5±3.1
1.1±1.0
3.3±2.0
2.5±2.0
0.25
1.78±0.39
1.47±0.33
1.48±0.85
1.56±0.44
1.73±0.89
0.96
Supplemental Table 3. Distribution of nerve size in proximal, middle, distal, and post
bifurcation location
Nerve size (mm)
0-<0.05
0.05-<0.10
0.10-<0.15
0.15-<0.20
0.20-<0.25
0.25-<0.30
0.30-<0.35
0.35-<0.40
0.40-<0.45
0.45-<0.50
≥0.50
Total
Proximal
73
171
78
61
31
18
16
6
3
2
8
467
Whole nerves (n=1339 nerves)
Middle
Distal
Post
53
47
70
203
167
61
97
66
15
55
30
7
22
26
8
18
14
6
18
10
7
5
8
0
7
7
1
4
3
3
8
4
0
490
382
178
Total
243 (16.0)
602 (39.7)
256 (16.9)
153 (10.1)
87 (5.7)
56 (3.7)
51 (3.4)
19 (1.3)
18 (1.2)
12 (0.8)
20 (1.3)
1517
Comparison between Proximal, Middle, Distal, and Post bifurcation based on each artery (n=8
renal arteries)
Proximal
Middle
Distal
P value for overall
All (n=8)
Post (n=8)
(n=8)
(n=8)
(n=8)
Mean Nerve
0.12±0.02
0.13±0.03
0.13±0.02
0.13±0.03
0.11±0.04
0.16
size (mm)
Repeated measures ANOVA was used.
Supplemental Figures and Figure legends
Supplemental Figure 1.
Supplemental Figure 1. Representative images of nerve size measurements. Renal artery and
peri-arterial nerves (A: H&E stain, B, C: neurofilament protein (NFP) stain). Panel C shows the
methods of nerve size measurement. Nerve’s short diameter was measured as the nerve size.
Supplemental Figure 2.
Supplemental Figure 2. The correlation between the number of nerves identified by NFP stain
and those identified by H&E stain is shown. Excellent correlation is demonstrated (r=+0.92,
P<0.0001) from 32 co-registered sections.
Supplemental Figure 3.
Supplemental Figure 3. The presence of sympathetic nerve fibers within kidney tissue. Panel A
is positive control; a nerve fascicle is present around the adrenal tissue. Red channel: TH; Green
channel: CGRP. Panel B shows a nerve fascicle in the kidney hilum. Abundant TH positive
fibers and few CGRP positive fibers are observed. Panel C shows nerve fibers (red) surrounding
an arteriole (A) within the kidney tissue. Panel D shows nerve fibers around renal tubules (T).
TH positive staining is predominant, but rare CGRP positive (arrow) nerve fibers are observed.
Panel E shows nerve fascicles around a renal tubule (T) and glomeruli (G). Panel F is highpower image of the white boxed area in panel E. TH positive staining is predominant with rare
CGRP positive (arrow) nerve fibers seen.