Vasoactive intestinal peptide-immunoreactive nerves in the rat kidney.

THE ANATOMICAL RECORD 219:193-203 (1987)
Vasoactive Intestinal Peptide-Immunoreactive
Nerves in the Rat Kidney
DAVID S. KNIGHT, JOHN A. BEAL, ZHI PING YUAN, AND TIMOTHY S. FOURNET
Department of Anatomy, School of Medicine, Louisiana State University,
Shreveport, LA 71130
ABSTRACT
An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was
used to visualize vasoactive intestinal peptide-immunoreactive (VIPD nerves in the
rat kidney. Rats were perfused with 4% paraformaldehyde or 2% paraformaldehyde
+ 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After
24-48 hours, the kidneys were sectioned with a Vibratome at 200 or 300 pm and
incubated in the primary antiserum for 18 hours at room temperature. A sparse
plexus of VIPI nerves innervates the rat renal calyx. Some VIPI nerves innervate
interlobar arteries and each succeeding segment of the arterial tree including afferent arterioles, but most innervate arcuate and interlobular arteries. VIPI axons do
not innervate each arcuate artery or each interlobular branch of an arcuate artery
with equal density. Although some axons follow each interlobular branch, most form
a dense plexus on only one or two branches. Therefore, most VIPI nerves in the rat
kidney innervate a restricted segment of the renal arterial tree. These nerves may
be efferent and may selectively dilate arcuate and smaller arteries, or they may be
afferent and may sense local changes in mechanical or chemical parameters.
The rat kidney is innervated by both sensory and
postganglionic autonomic nerves in which several neuroactive substances have been identified. Catecholamine-containing nerves form a uniformly dense plexus
on the arteries and also innervate the veins of the rat
kidney Gjungqvist and Wagermark, 1970).These nerves
are acetylcholinesterase-positive,
but there probably are
no intrarenal cholinergic nerves (Barajas et al., 1976).
Intrarenal nerves immunoreactive for substance P,
vasoactive intestinal peptide and calcitonin gene-related
peptides have been reported (Barajas et al., 1983; Ferguson and Bell, 1985; Su et al., 1986), but the relative
densities of peptidergic nerves on different parts of the
arterial tree have not been thoroughly studied. Whether
each peptide is synthesized by a separate nerve population or coexists with other peptides or with catecholamine, and whether each is contained in sensory or
autonomic nerves are points of interest that require
further study.
Immunohistochemical methods have been used to visualize vasoactive intestinal peptide (VIP)in both central
and peripheral neurons, including those that innervate
the heart and blood vessels where it is released by nerve
stimulation and probably causes vasodilation (Della et
al., 1983; Eklund et al., 1979; Heistad et al., 1980; Jarhult et al., 1980; Shimizu and Taira, 1979). Although
several workers failed to detect VIP-immunoreactive
(VIPI) nerves in pig, cat, rat, mouse, and guinea pig
kidneys (Alm et al., 1980; Larsson et al., 1977; Uddman
et al., 1981), Hokfelt et al. (1978) showed that VIPI
nerves innervate arteries in the guinea pig renal cortex,
and Forssman et al. (1982)found such nerves in kidneys
of the guinea pig, rat, cat, dog, pig, and Tupaia and
0 1987 ALAN R. LISS, INC.
noted that intrarenal distribution is subject to species
variation. Barajas et al. (1983)visualized VIPI terminals
in dog and rat kidneys. Most of these terminals innervate large arteries in the inner cortex and juxtamedullary region, but there are, in the dog kidney, some
terminals near glomeruli or the origins of arterioles.
Della et al. (1983) in a survey of VIPI nerves associated
with the guinea pig cardiovascular system found a
sparse-to-moderate number of terminals on the renal
artery, but few fibers on the renal vein and concluded
VIPI terminals are likely to be efferent and to effect
vasodilation when stimulated. Renal arterial terminals
are of moderate density compared with the most densely
innervated arteries supplying the gut, brain, and reproductive organs. In the present study, rat kidneys were
serially sectioned and immunolabeled to allow visualization and mapping of intrarenal VIPI nerves. The purpose of this study is to visualize VIPI nerves selectively
in thick sections to reveal nerve terminal distribution
and to illustrate relative densities on specific parts of
the arterial tree.
MATERIALS AND METHODS
Twelve Wistar rats of either sex (244-410 g) were used
in this study. Two fixation protocols were tried. Kidneys
were fixed by transcardial perfusion with 200 ml of 0.1
M Sorensen’s buffer + 0.5% procaine followed by 500 ml
of either 4% paraformaldehyde or 2% paraformaldehyde
0.15% picric acid in cold Sorensen’s buffer. After removal, kidneys fixed in 4% paraformaldehyde were fixed
+
Received October 20, 1986; accepted May 8, 1987.
Fig. 1. Sparse plexus of VIPI nerves (arrows) on the renal calyx. Renal tubules
X256.
(RT),renal calyx (RC).
Fig. 2. Large perivascular nerve bundle (NB) (Zontaining several VIPI axons (arrows). Arteriole (A).
x256.
in cold fixative for 2 hours, and those fixed in 2% paraformaldehyde
0.15% picric acid in cold fixative overnight were then transferred to cold Sorensen’s buffer.
After 24-48 hours in cold buffer, the kidneys were sectioned with a Vibratome a t 200 or 300 pm and collected
in the buffer. Sections of some kidneys were treated with
0.01% sodium borohydride in water (Farr and Nakane,
+
1981). All sections were presoaked for 1-2 hours in 0.1
M Sorensen’s buffer containing 0.3%Triton X-100, 5%
bovine serum albumin, and 2.5% normal goat serum
(NGS) prior to incubation in primary antiserum. The
antiserum to vasoactive intestinal peptide (VIP, INC
39H2T) was diluted 1:2,000 in 0.1 M Sorensen’s buffer
containing 0.3% Triton X-100 and 1%NGS. Ten milli-
VIP-IMMUNOREACTIVE NERVES
4
Fig. 3. VIPI nerves innervating an arteriolar branch of an interlobar artery. Arteriole (A), interlobar
renal artery @A). ~ 2 5 6 .
Fig. 4. Camera lucida drawing of VIPI nerves innervating intrarenal arteries. Arcuate artery (ARC),
interlobular artery (INT), VIPI nerve terminals (T), glomerulus (GI. X42. Inset X3.2.
195
196
D.S. KNIGHT, J.A. BEAL, Z.P. YUAN, AND T.S. FOURNET
liters of the diluted antiserum was absorbed with 0.2
mg of rat kidney powder for 30 minutes at room temperature and filtered (0.2 pm Gelman) prior to use. Kidney
sections were incubated in the primary antiserum for 18
hours at room temperature, rinsed in buffer, and treated
with biotinylated goat anti-rabbit IgG, then the avidinbiotinylated horseradish peroxidase (HRP) complex (Hsu
et al., 1981) as prescribed in the Vectastain procedure
(Vector Laboratories, Burlingame, CA). Control sections
were incubated for the same time period in normal (nonimmune) rabbit serum or in specific primary antiserum
preabsorbed with 1 x lOP4M synthetic VIP then processed as above. HRP was visualized by reaction for 10
minutes in 0.05% diaminobenzidine in Sorensen’sbuffer
(pH 7.3) containing 0.01% H202. The free-floating sections were then rinsed in buffer, dehydrated in graded
ethanols, placed in xylene, and mounted on slides in
Permount. A Leitz OrthopladOrthomat microscope and
80A filter were used to photograph specifically stained
nerves. A Leitz Dialux microscope equipped with a
drawing tube was used to trace the nerves innervating
selected arteries. Arterial segments whose nerve terminals were drawn lay wholly within the 300 pm tissue
section so that drawings illustrate terminals around the
circumference of the vessel. Kidney sections from which
these articles were selected were photographed so that
the location, orientation, and approximate size of the
vessel could be illustrated.
RESULTS
Incubation in either specific primary antiserum or
nonimmune control antiserum results in nonspecific
binding of serum components to thick ascending and
descending limbs of the loops of Henle. This causes moderate background staining in the outer medullary zone,
whereas background staining in the inner medulla and
cortex is light. Nevertheless, only neural elements stain
intensely after the treatment described above. A sparse
plexus of vasoactive intestinal peptide-immunoreactive
(VIPI) nerves innervates the rat renal calyx (Fig. 1).
Collagenous cords extend from the renal pelvis into the
parenchyma, and some VIPI axons extend along and lie
parallel with these cords; however, few if any innervate
the surrounding tubules.
Some VIPI nerves innervate interlobar arteries and
each succeeding segment of the arterial tree including
afferent arterioles. These nerves do not innervate efferent arterioles, although some juxtamedullary arterioles
that divide to form medullary vascular bundles are innervated. There are VIPI nerves associated with most
intrarenal arteries.
Near the renal hilus, large perivascular nerve bundles
contain VIPI axons most of which remain within the
nerve bundles throughout this region without ending or
giving rise to axon collaterals (Fig. 2). These axons do
not form compact VIPI nerve bundles but are dispersed
throughout the more numerous axons not reactive for
VIP. Most intrarenal VIPI axons are periarterial, but
few such axons innervate interlobar arteries; these do
not form a regularly spaced pattern. Some axons leave
the main nerve bundles to innervate arteriolar branches
of interlobar arteries (Fig. 3 ) or to form a sparse, localized plexus on the outer surface of the media of an
interlobar artery.
Most VIPI axons innervate arcuate and interlobular
arteries (Figs. 4-8).Axons on some arcuate arteries are
evenly distributed (Figs. 4, 61,whereas those on other
such vessels are clustered near the points where smaller
arteries branch (Figs. 5, 7). Such terminal clusters extend onto some branches so that middle to distal segments of arcuate arteries and proximal segments of
interlobular arteries branching from them have the densest and most complex VIPI plexuses of any intrarenal
vessels (Figs. 4-7, 9). These arteries are from 0.1 to 0.3
mm in diameter in the perfusion-fixed rat kidneys examined in this study. VIPI axons do not innervate each
arcuate artery or each interlobular branch of an arcuate
artery with equal density. Although some axons follow
each interlobular branch, most form a dense plexus on
only one or two branches (Fig. 7). Two arcuate arteries
are illustrated in Figures 5 and 7. Innervation of the
two vessels differs in that VIPI axons in Figure 5 are
concentrated on the parent arcuate artery, whereas most
of those in Figure 7 innervate two interlobular branches
whose parent vessel has few axons. The more regular
innervation pattern of the arcuate and interlobular arteries illustrated in Figure 4 was less frequently seen.
Arterioles also branch directly from arcuate arteries,
and many such arterioles have more axons than larger
branches nearby (Figs. 6,10, arrows). Axons on sparselyinnervated arteries appear to ramify at branch points
(Figs. 4,8, arrows).
Distal parts of the arterial tree (afferent arterioles and
distal segments and smaller divisions of interlobular
arteries) are sparsely innervated by VIPI nerves. Most
VIPI axons end along afferent arterioles proximal to the
juxtaglomerular apparatus, but some give rise to terminals near the glomerular vascular poles (Figs. 4,7, 11).
Few VIPI terminals invade the tubules surrounding
small cortical arteries. Although no VIPI axons innervate efferent arterioles, some axons that appear to arise
from the nerve plexuses of nearby arcuate arteries enter
the outer stripe of the outer zone to innervate arteriolae
and venae rectae of some medullary vascular bundles
(Fig. 12).
DISCUSSION
Vasoactive intestinal peptide-immunoreactive nerves
innervate the rat renal calyx and intrarenal arterial
tree. Dolezel(1975)described the renal connective tissue
skeleton, a system of collagenous cords extending from
the renal pelvis into the parenchyma, and noted that it
is innervated by monoaminergic and acetylcholinesterase-containing nerves. He suggested that some of these
terminals are sensory and detect changes in pressure in
the renal pelvis or parenchyma. VIPI nerves associated
with this system are sparse and are oriented mainly
parallel with the parenchymal extensions. VIPI terminals innervate interlobar arteries and succeeding segments of the arterial tree including afferent arterioles.
Nerve plexus density on different parts of the arterial
tree varies such that most terminals innervate arcuate
and interlobular arteries, while arteries proximal and
distal to these vessels have fewer terminals. This contrasts with noradrenergic nerves, which, in the adult
rat, form a uniformly dense plexus on all intrarenal
arteries as far distally as the afferent arterioles with
some terminals innervating efferent arterioles and med-
VIP-IMMUNOREACTIVE NERVES
197
5
Fig. 5. Camera lucida drawing of VIPI nerves innervating intrarenal arteries. Arrow indicates nerve
terminals near points where smaller arteries branch. Arcuate artery (ARC). x42. Inset x3.2.
Fig. 6. Camera lucida drawing of VIPI nerves innervating intrarenal arteries. Arcuate artery (ARC),
arteriole (A). ~ 4 2Inset
.
~3.2.
ullary vascular bundles as well (Ljungqvist and Wagermark, 1970; McKenna and Angelakos, 1968; Cowen et
al., 1982; Dhall et al., 1986). Although there are some
VIPI terminals on most intrarenal arteries, they do not
uniformly innervate all vessels of the same size or type.
For example, this study shows that VIPI terminals form
a moderately dense plexus on the initial segments of
some interlobular arteries, but other such vessels are
only sparsely innervated, and some are devoid of any
such terminals. Some arteriolar branches of interlobar
arteries are innervated in preference to the parent artery. Many arteriolar branches of arcuate arteries also
198
D.S. KNIGHT, J.A. BEAL, Z.P. YUAN, AND T.S. FOURNET
ARC
8
Fig. 7. Camera lucida drawing of VIPI nerves innervating intrarenal arteries. Arrow indicates nerve
terminals near points where smaller arteries branch. Arcuate artery (ARC), interlobular artery (INT),
VIPI nerve terminals (T), glomerulus (GI. X42. Inset ~ 3 . 2 .
Fig. 8. Camera lucida drawing of VIPI nerves innervating intrarenal arteries. x432. Inset x3.2.
VIP-IMMUNOREACTIVE NERVES
Fig. 9. Plexus of VIPI nerves innervating an artery in the juxtamedullary renal cortex. Renal artery
(RA). x256.
Fig. 10. VIPI nerves innervating an arteriole (A) in the juxtamedullary renal cortex. Arcuate artery
(ARC), convoluted tubules (CT). ~ 3 0 7 .
199
200
D.S. KNIGHT, J.A. BEAL, Z.P. YUAN, AND T.S. FOURNET
Fig. 11. VIPI nerve terminals (arrows) near the vascular pole of a glomerulus (GI. ~ 4 0 0 .
Fig. 12. VIPI nerve terminals (arrows) innervating a medullary vascular bundle in the outer stripe of
the outer zone. Straight tubules (ST).X256.
VIP-IMMUNOREACTIVE NERVES
appear to receive more axons than larger branches
nearby, but this is less evident because of the generally
greater numbers of VIPI axons on the arcuate arteries.
VIPI neurons whose terminals innervate the rat kidney may be a population of cells separate from those
containing norepinephrine or other neurotransmitters.
Neither chemical nor surgical destruction of noradrenergic axon terminals reduces vascular VIPI nerve
plexus density in several organs (Costa and Furness,
1982; Della et al., 19831, and renal arterial noradrenergic and VIPI nerves have different growth patterns
during development (Della et al., 1983). While there is
evidence for coexistence of VIP and acetylcholine in
some neurons (Lundberg et al., 1979a,b),many cholinergic nerves do not contain VIP (Lundberg et al., 19821,
and it is doubtful whether there are any cholinergic
nerves in the rat kidney (Barajas et al., 1976; DiBona,
1982). There are multiple peptides in various combinations in some sensory and autonomic neurons (Dalsgaard et al., 1983; Leah et al., 1985; Tuchscherer and
Seybold, 1985). Substance P-immunoreactive nerves innervate the rat kidney, but it seems unlikely that this
peptide and VIP coexist in intrarenal nerves, as VIP1
nerve distribution differs from that of substance P-immunoreactive nerves, most of which innervate interlobar vessels in the rat (Ferguson and Bell, 1985). The
distribution pattern of calcitonin gene-related peptideimmunoreactive nerves in the urinary system is similar
to that of substance P (Su et al., 1986).
Most evidence suggests that VIPI terminals originate
in autonomic ganglia and that VIP is released by nerve
stimulation in several peripheral organs where it relaxes vascular and nonvascular smooth muscle and excites secretory epithelial cells (Dalsgaard et al., 1983;
Elfvin, 1980;Fahrenkrug, 1982;Schultzberg et al., 1980).
There are VIPI cell bodies in spinal and nodose ganglia
and terminals in the superficial dorsal horn so that VIP
may be a primary afferent neurotransmitter concentrated in the sacral spinal cord (Loren et al., 1979;Lundberg et al., 1978; Honda et al., 1983; Kawatani et al.,
1983; Salt and Hill, 1983). Dun (1983), on the basis of
the origin and termination of VIPI nerves in the guinea
pig, suggested that VIPI terminals in the inferior mesenteric ganglion are sensory and mediate reflex modulation of ganglionic transmission in response to
stimulation of gut mechanoreceptors (Dalsgaard et al.,
1983).There are VIPI terminals in the cat carotid body,
but whether they are sensory or autonomic is unknown
(Lundberg et al., 1979a).
Several workers have shown that VIPI nerves are
vasodilatory, as VIP is released by nerve stimulation
and the exogenous peptide dilates many blood vessels
(Fahrenkrug, 1979; Lundberg et al., 1982; Said, 1982).
VIP infused into the renal artery increases renin secretion, but the mechanism of action is unknown (Porter
and Ganong, 1982; Rosa et al., 1985). It appears to be
independent of renal baroreflex or macula densa mechanisms, renal arterial dilation or changes in plasma
potassium concentration (Porter and Ganong, 1982;Rosa
et al., 1985). VIP appears to act directly on juxtaglomerular cells to increase renin secretion, and the effect
may be, in part, prostaglandin-mediated (Calam et al.,
1982). VIP influences the hypothalamus and modulates
the secretory activity of several endocrine glands, suggesting multiple roles for VIP in hormonal control sys-
201
tems (Hakanson et al., 1982; Polak and Bloom, 1982).
Increased solute excretion after VIP infusion into the
renal artery in the absence of significant changes in
total renal vascular resistance, perfusion flow rate, or
glomerular filtration rate suggests a direct effect on
renal tubular reabsorption (Rosa et al., 1985).Failure to
increase phosphate excretion suggests that the site of
action is distal to the proximal tubule, but the small
number of vascular bundles in which VIPI terminals
were identified in this study would not seem to support
a significant role for medullary parts of the nephron in
tubular responses to neurally released VIP. On the other
hand, there are few VIPI terminals near other segments
of the nephron, so that any tubular effects of VIP released by renal nerve stimulation may involve some
indirect mechanism.
The distribution of VIPI terminals observed in the
present work raises two questions. Why do most intrarenal VIPI terminals innervate a restricted region of the
arterial tree, and why are some arcuate and interlobular
branches relatively well innervated whereas others have
few terminals? If these nerves are autonomic, evidence
suggests that they release VIP, which relaxes smooth
muscle cells or stimulates cell secretion. Intrarenal VIPI
terminal distribution shown in this paper would apparently limit the principal effects to some segments of
arcuate and interlobular arteries. However, peptides are
effective in low concentration, are not recaptured by
nerve terminals, and have a long time course of action
postsynaptically so that sustained release may increase
VIP concentration in renal arterial blood by diffusion
through the vascular wall as with other neurally released substances (Bennett, 1972; Bradford, 1986) and
may cause effects downstream directly or by modulation
of catecholamine release or other mechanisms (Bradford, 1986; Krieger, 1983). Noradrenergic nerves innervate renal arterioles, juxtaglomerular apparati, and
some tubules but probably are not involved in the renin
response to VIP (Calam et al., 1982). It is evident from
the present study and that of Barajas et al. (1983) that
VIPI axons are dispersed throughout some large and
small intrarenal nerve bundles in which interaction with
noradrenergic axons is possible. These or other mechanisms may, in part, regulate arteriolar smooth muscle
tone, renin release, and renal tubular transport in response to neurally released VIP, as VIPI terminals do
not innervate most arterioles and tubules in the rat
kidney.
Intrarenal sensory nerves are both mechanoreceptive
(Astrom and Craaford, 1967)and chemoreceptive (Recordati et al., 19821, but data on sensory receptor distribution and ultrastructure and neurotransmitter synthesis
are minimal (Barajas and Wang, 1978; DiBona, 1982;
Kuo et al., 1984). Unilateral stimulation of these receptors may cause contralateral renal vascular constriction,
pressor or depressor responses systemically, or a change
in nerve activity or catecholamine content in hypothalamic nuclei involved in body sodium and water balance
(Aars and Akre, 1970; Calaresu and Ciriello, 1981a,b;
Calaresu et al., 1976; Colindres et al., 1980; Katholi et
al., 1983;Recordati et al., 1982).Ifintrarenal VIPI nerves
are sensory, the partial innervation of arcuate and interlobular arteries in the inner cortex and juxtamedullary
zone may suggest that sensory input originating at these
strategic points in the arterial tree is adequate to moni-
202
D.S. KNIGHT, J.A. BEAL, Z.P. YUAN, AND T.S. FOURNET
tor blood flow, blood pressure, or other parameters involved in effecting these responses.
In the rabbit renal artery, nerve growth is continuous
from birth to age 6 months after which time a gradual
nerve loss occurs even though the proportion of varicosities to nerve fibers increases (Cowen et al., 1982).Innervation density of guinea pig renal arteries gradually
decreases postnatally (Gallen et al., 1982). There is no
evidence as to whether intrarenal VIPI axon terminals
in rats form static plexuses or are involved in the kind
of nerve growth or regression that occurs in these species, but either process could explain the nonuniform
distribution of axon terminals (Dhall et al., 1986).
Most VIPI nerves in the rat kidney innervate a restricted segment of the renal arterial tree. These nerves
may be efferent and may selectively dilate arcuate and
smaller arteries, or they may be afferent and may sense
local changes in mechanical or chemical parameters.
ACKNOWLEDGMENT
This work was supported by NIH grant HLB34431.
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