Pflugers Arch - Eur J Physiol (2013) 465:25–37
DOI 10.1007/s00424-012-1126-7
INVITED REVIEW
Regulation of renin secretion by renal juxtaglomerular cells
Ulla G. Friis & Kirsten Madsen & Jane Stubbe &
Pernille B. L. Hansen & Per Svenningsen & Peter Bie &
Ole Skøtt & Boye L. Jensen
Received: 18 May 2012 / Revised: 2 June 2012 / Accepted: 6 June 2012 / Published online: 26 June 2012
# Springer-Verlag 2012
Abstract A major rate-limiting step in the renin–angiotensin–
aldosterone system is the release of active renin from
endocrine cells (juxtaglomerular (JG) cells) in the media
layer of the afferent glomerular arterioles. The number
and distribution of JG cells vary with age and the
physiological level of stimulation; fetal life and chronic
stimulation by extracellular volume contraction is asso-
ciated with recruitment of renin-producing cells. Upon
stimulation of renin release, labeled renin granules “dis-
appear;” the number of granules decrease; cell mem-
brane surface area increases in single cells, and release
is quantal. Together, this indicates exocytosis as the
predominant mode of release. JG cells release few per-
cent of total renin content by physiological stimulation,
and recruitment of renin cells is preferred to recruitment
of granules during prolonged stimulation. Several endo-
crine and paracrine agonists, neurotransmitters, and cell
swelling converge on the stimulatory cyclic AMP
(cAMP) pathway. Renin secretion is attenuated in mice
deficient in beta-adrenoceptors, prostaglandin E2–EP4
receptors, Gsα protein, and adenylyl cyclases 5 and 6.
Phosphodiesterases (PDE) 3 and 4 degrade cAMP in JG
cells, and PDE3 is inhibited by cyclic GMP (cGMP)
and couples the cGMP pathway to the cAMP pathway.
Cyclic AMP enhances K+-current in JG cells and is
permissive for secretion by stabilizing membrane potential
This article is published as part of the special issue on the Renin-
Angiotensin System.
U. G. Friis : K. Madsen : J. Stubbe : P. B. L. Hansen :
P. Svenningsen : P. Bie : O. Skøtt : B. L. Jensen (*)
Department of Cardiovascular and Renal Research,
Institute of Molecular Medicine, University of Southern Denmark,
J. B. Winslowsvej 21, 3,
5000, Odense C, Denmark
e-mail: bljensen@health.sdu.dk
far from threshold that activates L-type voltage-gated calcium
channels. Intracellular calcium paradoxically inhibits renin
secretion likely through attenuated formation and enhanced
degradation of cAMP; by activation of chloride currents and
interaction with calcineurin. Connexin 40 is necessary for
localization of JG cells in the vascular wall and for pressure-
and macula densa-dependent suppression of renin release.
Keywords Cyclic AMP . Juxtaglomerular cells . Renin
secretion
Introduction
Renin secretion has been the topic of comprehensive
reviews in 2010 [15] and 2011 [57]. In the present review,
focus will be on discoveries that have added significant
knowledge on the cellular mechanisms that govern secretion
of active renin from renin-producing cells in the afferent
glomerular arterioles. Active renin of renal origin deter-
mines plasma and, hence, extracellular fluid renin levels.
Active renin in plasma and tissues disappears after bilateral
nephrectomy [27]. A nonsecreted intracellular form and a
secreted form of active renin have been detected in the brain,
but its deletion did not alter blood pressure or circulating
renin [134]. In human plasma, prorenin or other inactive
forms of renin constitute about 80–90 % of circulating total
renin [82]. Large amounts of prorenin can be infused in
monkeys with no apparent physiologic effect on hemody-
namic and kidney parameters [71]. Thus, experimental evi-
dence for the existence of a physiologically significant
conversion of prorenin to active renin in the circulation is
not available, and the renin-producing cells of the afferent
glomerular arteriole in the kidney are the only source of
active renin that respond to extracellular volume/blood
26
pressure-related stimuli. These cells are often called juxta-
glomerular (JG) cells referring to the typical location in
physiological sodium-replete states, but renin-producing
cells can extend far beyond the JG area. JG cells display
dual smooth muscle cell-like characteristics and endocrine
characteristics. Within JG cells, cathepsins, colocalized with
prorenin in the secretory granules, have been suggested to
activate prorenin to renin. Data from gene-targeted mice
show that at least cathepsin B is not crucial for this activa-
tion [79], and the prorenin-to-renin-processing enzyme has
not yet been clearly identified as reviewed by Gross et al.
[40]. The number of JG cells varies considerably with
individual’s age and extracellular fluid volume status. Con-
ditions of extracellular volume depletion, e.g., low NaCl
intake [122], alter the number of differentiated renin-
producing cells by upstream differentiation of media cells
to renin-secretory cell, a phenomenon referred to as
recruitment.
Morphology of renin-positive cells
Fully differentiated JG granular cells have a large nucleus, a
hypertrophic rough endoplasmic reticulum, and distinct
Golgi apparatus. Two main types of secretory granules can
be discerned in the cytoplasm. Large electron-dense mature
granules contain primarily active renin, angiotensin (ANG) I
and ANG II peptides, and cathepsins. In addition, smaller,
more electron-lucent protogranules leave the Golgi appara-
tus and contain active renin and prorenin [47]. The renin-
secretory granules have several features in common with
lysosomes: an acid pH, a content of typical lysosomal
enzymes like acid phosphatases, and the ability to take up
extracellular particulate matter and degrade cellular constit-
uents like internalized membrane material [124]. Renal re-
nin gene expression and active renin content show
developmental regulation and peak around the time of birth
most clearly established in rat, when a large part of the renal
arterial vasculature is renin positive [39]. As the individual
matures into adulthood, the localization of renin expression
regresses to include only the most distal or juxtaglomerular
part of the afferent arterioles, but maintains the capacity to
switch to a fetal pattern of increased renin cell distribution.
Recruitment of renin-positive cells has been observed after
pharmacological inhibition of renin, ANG II type 1 (AT1)
receptors, angiotensin-converting enzyme (ACE), and after
targeted disruption of genes encoding components in the
cascade from angiotensinogen over renin to aldosterone
synthase, mineralocorticoid receptor, and Na+ transporters
within the thick ascending limb extending to the collecting
ducts (e.g., [29]). The molecular and cellular mechanisms by
which the changes in cell phenotype associated with recruit-
ment occur are currently being unraveled.
Pflugers Arch - Eur J Physiol (2013) 465:25–37
Mechanisms that govern JG cell differentiation
Renin promoter-driven expression of intravital dyes has
been used to track renin-expressing cells and their descend-
ants and has shown that the renin lineage of cells differen-
tiate into other cell types in adult life (arteriolar smooth
muscle, but also intra- and extraglomerular mesangial cells,
proximal tubule cells, and Bowman’s capsule cells), which,
however, retain the capacity to synthesize renin and convert
to a renin cell phenotype upon chronic stimulation of the
system [114]. Thus, recruitment appears to be associated
with those cells that expressed renin during embryonic/early
postnatal life and suggest that only these cells retain the
ability to convert into a renin-producing phenotype. Of
interest, renin-lineage cells were also observed in the testis,
adrenal gland, stomach, and thymus [114]. By which cellu-
lar signals do these shifts in phenotype occur? The
microRNA-processing enzyme Dicer was deleted specifical-
ly in the JG cell lineage and resulted in a phenotype with
fewer renin-positive cells, lower renin mRNA expression,
lower plasma renin concentration and blood pressure, and
progressive decline in renal function typically observed after
deletion of renin–angiotensin system components from birth
[115]. Thus, microRNAs, which are small noncoding
RNAs, appear essential for differentiation and/or mainte-
nance of JG cells. In particular, miR-330 and miR-125b-5p
control the smooth muscle phenotype of the renin cell
lineage [77]. A phenotype somewhat similar to that ob-
served after JG cell-specific deletion of Dicer was observed
by deletion of the notch receptor transcription factor RBP-J
[14, 115]. After FACS sorting of labeled JG cells, the first
comprehensive “map” of renin cell-specific gene expression
pattern was presented. Pools of highly renin cell- and
smooth muscle cell-specific genes belonging to transporters,
enzymes, and transcription factor families were uncovered,
and of specific interest, pathways regulated by calcium and
cyclic AMP (cAMP) were identified [8]. Is there a common
stimulus for the recruitment phenomenon at the systemic
level? ANG II AT1A receptor deletion in mice combined
with a low-salt diet uncovers a much more marked recruit-
ment compared to AT1A-intact mice and is paralleled by a
more marked decrease in blood pressure. When blood pres-
sure was kept high by inhibition of nitric oxide synthase or
by infusion of alpha-adrenergic agonists during low dietary
NaCl intake in AT1A−/− mice, the renin cell recruitment was
attenuated, suggesting that pressure is a more important
signal compared to low transepithelial NaCl transport rate
[74]. Recruitment of JG cells during fetal life depends on
intact beta-adrenoceptors [81] and on the adenylyl-
stimulating Gsα protein [81], which indicates that renal
nerves, together with pressure, might have separate roles
for recruitment. The observation that salt depletion or ACE
inhibition of hydronephrotic mice with no macula densa
Pflugers Arch - Eur J Physiol (2013) 465:25–37
also [4] leads to renin cell recruitment, and increased renin
tissue content supports that pressure/vascular wall signals
are sufficient to regulate renin cell recruitment in the com-
plete absence of signals from the tubular epithelium.
Mode of renin release
The renin storage granules are evenly distributed in the
cytoplasm, and there is no apparent morphological polari-
zation of the release process towards the vascular lumen or
renal interstitium. Thus, renin is released to plasma and a
certain fraction of this renin is filtered at the glomerulus and
probably taken up by the proximal tubule [72]. Some renin
is secreted into the interstitial compartment, which is
reflected in a high concentration of active renin in renal
lymph [133].
Morphology of renin release
The renin release process exhibits features of exocytosis that
include close contact between mature granules and the plas-
ma membrane, membrane-limited omega-shaped recesses,
and local changes of the granular matrix at the site of
membrane alignment; whereas, fusion areas, where the
two trilaminar membranes have merged, are an extremely
rare observation, and plane fusion between the two mem-
branes has not typically been observed in ultrathin sections
[121–123]. After intense stimulation of renin release in
mice, a contact area is observed between the renin storage
granule membrane and the plasma membrane, which is
limited to a small protrusion of the granule [120, 121].
Within the protrusion, the granule matrix is less dense and
a local increase of granule volume becomes apparent [120,
121]. In vitro, similar changes in renin granule morphology
were observed when renin secretion was stimulated. The
granules were often clustered close to the plasma mem-
brane, and sites of putative fusion were observed [120]. A
frequent observation during intense stimulation of renin
secretion is the emergence of large membrane-bounded
omega-shaped membrane recesses, which label for renin
protein and contain modified granule matrix. These recesses
are closely associated with the plasma membrane [3, 84,
121]. Because the plasma membrane-associated structures
label for renin, they are considered to represent a late stage
of the release process, when most of the granule content has
been released. The tracking of renin protein from early
protogranules through electron-dense mature granules to
membrane-bound recesses strongly suggests that the
recesses represent empty granules that have undergone exo-
cytotic membrane fusion. Moreover, in immunohistochem-
ical studies, renin immunopositivity was not typically
observed within the cytoplasmic matrix, which speaks
27
against intracytoplasmic solubilization as a significant path-
way for renin release [3, 121]. The data indicate that the
initial fusion pore and small contact point are rapidly en-
larged, and a more long-lived conformation is established
with folded or endocytosed membrane material. Quantita-
tive stereological counting of renin granules in rat kidneys
was used to test the hypothesis that exocytosis would be
expected to result in fewer granules. Acute stimulation of
renin release in rats with a chronically prestimulated renin–
angiotensin–aldosterone system (RAAS) (by combined
ACEI and low NaCl) yielded a significant reduction in renin
granule numbers [92]. This reduction in granule number
agrees with the concept that mature granules fuse with the
cell membrane and empty their contents by exocytosis and
thus “disappear.” Stereological counting of renin granules at
the electronmicroscopic level in the spontaneous mutant of
the C57 strain “beige” mutant mice with impaired systemic
granule biogenesis showed that this mice strain had 1–2
renin granules per cell with multiple fingerlike processes
and a significantly lower plasma renin concentration than
control mice [50]. Each granule was about 300 times larger
compared to a control mouse renin granulum [50]. If it is
assumed that granules contain an amount of renin that
corresponds to the size of the granule, the observation of
plasma renin concentrations in low–normal range suggests
that parts of granules or granule contents can be released or
that renin is released very slowly from the granule matrix.
Renin release mode-functional data
Collection of superfusion fluid with high time resolution
from single rat afferent arterioles showed that renin is re-
leased in quanta with a renin content in each quantum,
which corresponds to the calculated content of one renin
granule [117]. The concept of exocytosis is supported by
data obtained with the whole-cell patch-clamp technique by
which cell capacitance, Cm, an accepted measure for cell
membrane surface area, can be recorded continuously dur-
ing maneuvers predicted to stimulate renin release [30]. A
range of extra- and intracellular factors known to alter renin
secretion by classic biochemical measurement of enzymic
renin activity (cAMP, cell swelling, and isoprenaline) in-
creased Cm 5–10 % over minutes in single JG cells compat-
ible with addition of surface membrane and, thus, fusion of
renin granules [30]. Real-time imaging of JG cell granules
in an in vitro system with high resolution demonstrated
rapid “disappearance” of granules compatible with fusion
and release of granule contents [89].
Renin secretion from isolated perfused kidneys also dem-
onstrate very transient renin responses upon osmotic stimuli
that could be repeated after a lag period of minutes and,
secondly, that the peak transient responses depended on the
prestimulatory level of renin secretion [66]. These findings
28
all fit the concept that there exists a “readily releasable pool”
of renin granules available for prompt exocytosis. The iso-
lated perfused kidney data further support that the pool of
readily releasable granules is inhibited by calcium-dependent
stimuli and supported by the cAMP pathway [66]. Of note,
supraphysiological stimulation of renin release from single JG
cells led to significant decrease in Cm and, hence, cell mem-
brane area, which suggests either regular “loss” or by well-
known “compensatory” membrane retrieval [30]. After mod-
erate stimulation of renin release and prolonged recording
time, an increase of Cm was followed by a compensatory
decrease in Cm to the prestimulatory level [31]. If this reflects
membrane retrieval, it would be compatible with the observa-
tion that renin cells take up external markers and internalize
particles into renin granules that behave like lysosomes; ex-
ogenous tracers, like ferritin, reach juvenile renin granules
without labeling the Golgi complex, and data show a large
membrane turnover in renin granules [123, 124]. What con-
trols intracellular renin granule trafficking? Targeted deletion
of “dense core vesicle proteins,” which are granule-associated
proteins known from pancreatic beta cells, leads to lower
levels of renin expression and plasma renin concentra-
tion [55]. However, since these proteins do not coloc-
alize with renin, the lower renin levels were interpreted
as an indirect effect. Data showed that the deleted dense
core vesicle proteins impaired catecholamine vesicle
processing in mice and thereby attenuated sympathetic
stimulation of renin [55]. The vesicle-associated mem-
brane protein (VAMP) 2 and VAMP3 were recently
shown in JG cells and knock-down in vitro of VAMP2,
but not VAMP3 attenuated cAMP-mediated renin release
from isolated cells [78]. Further studies are warranted
along this line.
Renin stores, granule numbers, and renin release
There is a constant, constitutive release of prorenin from JG
granular cells, probably through vesicles that are different
from mature storage granules [41]. Rapid changes in plasma
active renin are not reflected by similar rapid changes in
plasma prorenin, which argues against cosecretion of active
renin and prorenin [3]. The rate of prorenin release primarily
reflects the rate of renin synthesis [91]. What is the physi-
ological significance of renin cell differentiation during
chronic stimulation of the system? Is renin release depen-
dent on renin cell recruitment because absolute stores of
active renin are limited or because the release process can-
not recruit stored renin granules to an extent that suffices to
meet the circulatory demand in situations with extracellular
volume depletion or low renal perfusion pressure? The first
question can be easily answered since kidney tissue renin
content suffices to maintain circulating levels for hours to
Pflugers Arch - Eur J Physiol (2013) 465:25–37
days. In rats with chronic stimulation of the RAAS by
enalapril and low salt, a 36-fold elevated level of circulating
active renin was observed [92]. Upon acute stimulation of
renin secretion by lowering perfusion pressure to 40 mmHg
for 5 min, there was a significant and fairly similar twofold
increase in plasma renin concentration in both control and
prestimulated rats. Thus, a similar order of magnitude of
relative stimulation in response to acute stimulation al-
though the prestimulatory absolute levels of plasma and
tissue renin differed markedly. In mice with constitutively
lower renin stores and lower JG cell number (JG cell-
specific Gsα−/−, COX-2−/−, and dense core vesicle pro-
tein−/−), similar observations have been made. The absolute
level of renin secretion (delta plasma renin) in response to
acute stimuli (furosemide, hydralazine, and isoproterenol
boli) corresponds to the preexisting level of renin expres-
sion, while fold changes are rather similar [17, 53, 55].
Thus, across species and kidney renin levels, there is a
significant correlation between the number of renin-
producing cells and the absolute release potential. In total,
this results in a rather constant relative release potential.
In agreement, stereological quantification of granule-
containing volume of afferent arterioles in rat kidney showed
that the volume of the afferent arteriole that contained renin
did not decrease despite decrease in granule numbers [92].
This observation suggests that a limited pool of renin granules
is released from each JG cell instead of complete depletion of
individual JG cells. Recruitment of stored granules beyond
this acute release potential is limited. The capacitance meas-
urements of surface membrane changes in JG cells were
compared to granulum size and granulum pool size, and this
has yielded a rough estimate of the number of stored granules
that are released at ca. 5 % upon standard stimuli [30]. The
mouse afferent arteriole (NMRI strain) contains on average
1,900 granules/arteriole by stereological counting on standard
rodent diet [50]. In the rat, stereological counting yielded on
average 445 renin-secretory granules per afferent arteriole on
a standard diet [92]. Chronic stimulation increased the number
20 times to ca. 9,000 granules [92]. Calculations have sug-
gested that, in the rat, the release of one renin granulum per
afferent arteriole per 5 min maintains basal PRC [117]. A
similar estimation has yielded one each 20 min per arteriole
in the mouse [121], and with ca. 1,900 granules per arteriole in
mice, this provides theoretically the mouse with sufficient
renin granules for 26 days and the rat with a smaller potential.
So despite the large stores of active renin within the kidney,
the full secretory capacity of preexisting JG cells is not
exploited. Recruitment of more JG cells is preferred over
enhanced intracellular granule recruitment processes. The
evolutionary background for this dominant negative regula-
tion of renin secretion could be the danger of evoking a
hypertensive crisis by uncontrolled renin release. These obser-
vations contribute to the understanding of how it is possible
Pflugers Arch - Eur J Physiol (2013) 465:25–37
for the renin–angiotensin system to be involved in short-term
regulation of blood pressure via acute effects on peripheral
resistance, while at the same time, the system can be involved
in the long-term regulation of blood pressure by its effects on
sodium and water homeostasis. After chronic stimulation of
the system, the circulating renin and ANG II concentrations
are high. In this situation, the ANG II receptor expression is
downregulated in the peripheral blood vessels, whereby, a
hypertensive crisis is avoided. The renin–angiotensin system
maintains its ability to regulate short-term blood pressure
(e.g., after changing position), also in a situation with massive
chronic stimulation, because the relative increase in renin
release is the same as in the control situation.
Membrane potential and its influence on renin secretion
Stimulus–secretion coupling often depends on electrical sig-
nals that initiate the process. JG cells in situ are polarized (−50
to −70 mV) [9–12, 26], and membrane potential influences the
release process. Bührle et al. observed that addition of agonists
with intracellular coupling to cAMP had no effect on mem-
brane potential (Vm), while calcium-dependent agonists led to
depolarization [9, 11]. It was concluded that stimulation of
renin release was independent of membrane potential, while
inhibitory signals could depend on membrane depolarization.
The first whole-cell patch clamp study on JG cells in isolated
mouse afferent arterioles showed depolarization by inhibition
of inward-rectifier K channels (KIR) after addition of ANG II
[63]. Subsequent studies confirmed that JG cells express KIR
channel subunits [70]. At normal resting membrane potential
in JG cells, the dominant potassium conductance is through
calcium- and cAMP-sensitive BKCa channels (KCa1.1, ZERO
variant) and, to a lesser degree, through voltage-gated KV
channels [33]. Setting Vm in JG cells at different levels did
not per se induce changes in Cm and, hence, exocytosis [33].
Depolarization in one, but not other studies showed activation
of classical voltage-gated calcium currents [33, 64, 103]
which could be blocked by selective L-type antagonists [33,
64, 103]. Both RNA and protein for the L-type, voltage-gated
calcium channel (CaV) 1.2 was observed in single JG cells
[33]. A functional role of voltage-gated calcium channels was
indicated by the observation that depolarizing the membrane
potential inhibited cAMP-mediated increases in cell mem-
brane capacitance in an L-type channel blocker-sensitive
way [33]. So what is the integrated role of Vm in
stimulus–secretion coupling in JG cells? JG cells in the
vascular wall are quite polarized −50 to −60 mV and thus at
prevailing conditions far from levels of Vm that activate
the L-type calcium current which is maximal at
+10 mV. It is likely that the hyperpolarizing influence
of cAMP-coupled agonists through KCa1.1, ZERO vari-
ant is permissive and serves to stabilize membrane
29
potential in a range far from the threshold for activation
of voltage-gated calcium channels. On the other hand,
significant depolarization (as e.g., after ANG II) may
depolarize JG cells to level of Vm that activate L-type
calcium channels and attenuate cAMP-mediated stimula-
tion of renin release.
Intracellular signaling pathways that control exocytosis
in juxtaglomerular cells
Cyclic AMP pathway
Several physiologically relevant para- and endocrine agonists
and neurotransmitters coupled to activation of adenylyl cy-
clase stimulate renin release and increase cAMP in JG cells.
Mice with deficiency in the adenylyl cyclase activating Gsα
protein in JG cells display lower renin stores and plasma renin
concentration [17]. There is an attenuated renin secretion
response from isolated Gsα protein-deficient-isolated JG cells
to β1-adrenoceptor agonist isoproterenol and PGE2 [1, 17].
Adenylyl cyclases 5 and 6 catalyze cAMP formation in JG
cells [1, 86, 87] (Fig. 1). Phosphodiesterases (PDE) 3 and 4 are
expressed in renal vessels and JG cells [97] and use primarily
cAMP as a substrate. PDE3 and PDE4 degrade cAMP in JG
cells since selective PDE3 and PDE4 inhibitors stimulate
renin secretion in conscious rabbits and humans [20–22, 93]
and in isolated perfused rat kidney [61] (Fig. 1).
Cyclic GMP pathway and coupling to cAMP pathway
Agonists that raise intracellular cGMP in granular cells,
such as nitric oxide (NO) and atrial natriuretic peptide
[60], have been reported to have either an inhibitory [44]
or a biphasic [108] effect on renin release rate in vitro. In
vivo and within the isolated perfused kidney preparation, it
is a rather consistent finding that NO donors promptly
stimulate renin secretion [102]. NO donors depend on an
intact protein kinase A (PKA) pathway for stimulation of
renin release, which suggests an effect mediated by cAMP
rather than cGMP [61]. NO donors potently increase cGMP
in JG granular cells, but cAMP is also stimulated [108]. The
putative connection between the two pathways in the regu-
lation of renin secretion was established by discovery of
marked stimulation of renin secretion by pharmacological
block of PDE3 [20–22, 61]. PDE3A and PDE3B were
demonstrated in afferent arterioles [97], and while PDE3
degrades cAMP, it is inhibited by cGMP (Fig. 1). Sensitivity
towards PDE3 blockers and PDE3 expression was detected
also at the level of the single JG cell where intracellular
dialysis with cGMP-enhanced exocytosis as determined by
a cell capacitance increase dependent of protein kinase A
activity [32]. It is conceivable that at least part of the
30
stimulation of renin release in response to NO is caused by
inhibition of cAMP degradation.
Two cGMP-dependent protein kinases have been identi-
fied, and both are expressed by JG granular cells, in particular,
the cGMP-regulated protein kinase type II (cGKII) [36]. In
mice with targeted disruption of cGKII, cGMP-dependent
agonists are unable to inhibit renin secretion [131], while in
granular cells which have been transfected to overexpress
cGKII, there is marked inhibition of cAMP-stimulated renin
release [37]. Thus, the renin-secretory response to cGMP-
dependent agonists is determined by the dominance of the
cGKII pathway compared to the PDE3 pathway. Most data
suggest that PDE3 is constitutively expressed, whereas cGKII
is regulated [36], but at present, the significance of this is not
established. Altogether, cGMP has complex effects on renin
secretion, which are determined by the prevalence of two
mutually antagonistic target pathways for cGMP in stimu-
lus–secretion coupling (Fig. 1). At present, the data suggest
that the stimulatory pathway for cGMP depends on cAMP.
Fig. 1 Signaling pathways in the stimulus–secretion coupling for
renin in renal juxtaglomerular granular cells. Arrow indicates stimula-
tion and a line with foot indicates suppression. Cyclic AMP, cGMP, and
free ionized calcium are crucial second messengers in the pathways
that govern exocytosis of renin. Several agonists (e.g., PGE2, norepi-
nephrine, and PGI2), acting through specific receptors (R), converge on
Gsα protein-dependent activation of adenylyl cyclases (AC5 and 6)
and synthesis of cAMP. Protein kinase A is necessary for cAMP-
mediated stimulation of exocytosis measured as an increase of cell
capacitance (Cm). Moderate cell swelling occurs through aquaporin 1-
mediated water uptake and also leads to PGE2-EP2/4 receptor activa-
tion of the cAMP pathway. PKA increases K+ current though BKCa
channels (ZERO variant). This leads to hyperpolarization of membrane
potential (Vm), which is permissive for renin release by preventing
activation of voltage-gated calcium channels (CaV). Cyclic AMP is
degraded through phosphodiesterases 3 that is inhibited by cGMP, and
PDE1 that is stimulated by Ca2+. Cyclic AMP prevents liberation of
Pflugers Arch - Eur J Physiol (2013) 465:25–37
Calcium-regulated pathways
The paradoxical inhibitory role of calcium in renin release is
reviewed comprehensively by others in the current issue and
is, therefore, not detailed in the present review. What is
current status on mechanisms for this effect? Agonists that
raise [Ca2+]i in granular cells prevent the stimulatory action
of cAMP-dependent agonists on renin release [94]. Depo-
larization of the membrane potential prevents cAMP-
stimulated exocytosis in JG cells in a way sensitive to
calcium channel blockers [33]. These observations are com-
patible with an enhanced degradation or reduced formation
of cAMP by calcium in JG cells. Both adenylate cyclase and
cAMP phosphodiesterases exist in calcium-sensitive iso-
forms, and experimental evidence indicates that calcium-
stimulated PDE1 is present in JG cells and contribute to
the inhibition of renin release by calcium [85] (Fig. 1).
Calcium-inhibitable adenylyl cyclase V is present in JG
cells, and chelation of calcium increases cAMP formation
calcium from intracellular stores (ER). Intracellular cGMP increases
through soluble guanylyl cyclase and membrane receptor-activated
guanylyl cyclase activity through nitric oxide and atrial natriuretic
peptide. Cyclic GMP can either stimulate renin release by inhibition
of PDE3 or inhibit renin release through cGMP-regulated protein
kinase. Gi-coupled agonists like angiotensin II, extracellular calcium,
or endothelin cause an increase of intracellular free calcium concentra-
tion by release of calcium from intracellular stores and by membrane
depolarization (Vm) with subsequent activation of transmembrane cal-
cium influx through CaV. Membrane depolarization is caused by inhi-
bition of anomalous inward-rectifier K channel and by stimulation of
calcium-activated chloride channels. This could lead to coordinated
efflux of KCl and cell shrinkage. Calcium suppresses renin secretion
likely through serial activation of calmodulin and calcineurin and by
parallel inhibition of cAMP. Cyclic AMP degradation by PDE1 is
enhanced, and cAMP formation is inhibited through AC5/6
Pflugers Arch - Eur J Physiol (2013) 465:25–37
and renin release [1, 86] and JG cell membrane capacitance
[75]. Blockers of calmodulin enhance renin release and JG
cell membrane capacitance [25, 75]. Calcium is not likely to
control renin solely through cAMP, because release rate can
be changed without alterations in cAMP [65]. Calcium
could affect renin secretion by a direct interaction with ion
channels in the cell membrane. In fact, the most direct effect of
a rise in [Ca2+]i reported is activation of a chloride conduc-
tance [63, 103] (Fig. 1). A calcium–calmodulin-dependent
phosphatase, calcineurin, appears also to control exocytosis
of renin by yet undefined interactions as judged by sensitivity
of renin release from isolated JG cells to clinically well-
established blockers of calcineurin, e.g., cyclosporine [58,
75] (Fig. 1). The second messenger cyclic ADP-ribose
through Ca2+ signaling appears also to be a potential pathway
that exist at least in the renin-producing AS4-1 cell line [138].
Osmotic forces
Renin secretion from in vitro preparations exhibits a high
sensitivity to changes in extracellular osmolality such that a
modest, e.g., ~5–10 % reduction in the extracellular osmo-
lality, leads to rapid increases in renin secretion [28, 104,
127]. Changes in osmolality induced by nonpermeant sol-
utes are more efficient in inducing changes in renin release
than permeant solutes, which implies that it is the osmotic
effect rather than a specific ionic effect that causes the
change in renin release. JG cells with permeabilized cell
membranes retain the ability to release renin upon exposure
to a moderate decrease in osmolality, which indicates that
renin granules are osmotically sensitive [52]. In contrast, in
more complex models, i.e., the perfused kidney and in vivo
settings, an increase in osmolality stimulates renin secretion
[2, 66]. The extracellular osmolality in the JG area has been
suggested to vary depending on transepithelial NaCl trans-
port rate by the rather water impermeable macula densa
[88]. JG cell capacitance is rapidly increased by a decrease
in extracellular osmolality or by introduction of slightly
hyperosmotic solution into the cytoplasm [30]. Thus, cell
swelling, and not granule swelling, is necessary for the
stimulus–secretion response to hypoosmolality, at least in
single JG cells. The response depended on presence of the
water channel aquaporin 1 (AQP1) and on intact cAMP-
PKA signaling [34]. An autocrine loop was suggested based
on the dependence of cell swelling-induced renin release on
phospholipase A2 (PLA2) and COX-2 activities [34]. The
findings indicate that slight hypoosmotic extracellular fluid
leads to AQP1-mediated water influx in JG cells, PLA2/
COX-2/prostanoid-dependent cAMP formation, and activa-
tion of PKA, which promotes exocytosis of renin. The
difference between renin release responses to changes in
osmolality in vivo and in reduced in vitro settings is not
clear at present.
31
Cellular pathways for major systemic controllers
of renin
“Baroreceptor”
It was suggested by Tobian [125] and demonstrated by
Skinner et al. [116] that there existed a renal baroreceptor
by which arterial blood pressure negatively controlled renin
release. In the denervated, nonfiltering dog kidney, Blaine et
al. [5] demonstrated that pressure-regulated renin release
was intact. There is now agreement that the baroreceptor
mechanism resides in the renal vasculature itself. The baro-
receptor has been extensively characterized with respect to
“dose” (pressure)–response relationships [56] and calcium
dependence [100, 101]. Data suggest that pressure-sensitive
renin release is independent of voltage-gated calcium chan-
nels [101, 103], but dependent on extracellular calcium
[100]. It has been difficult to establish reproducible data
on baroreceptor function in vitro with isolated arterioles.
Flow appears to be required to obtain pressure-sensitive
renin responses with isolated renal microvessels [6, 96]. In
contrast, it has been shown that mechanical stretch leads to
significant inhibition of renin release and reduced renin
mRNA abundance in cultured rat granular cells [13]. In-
creased pressure leads to elevated intracellular calcium con-
centration in a dose-dependent way and sensitive to L-type
calcium channel blockers and cation channel blockers in JG
cells in situ within isolated murine arterioles [68]. Are
mediators involved in the response? Adenosine acting
through A1 receptors is necessary for high pressure-
induced suppression of renin secretion, while lowering of
pressure led to adenosine-independent stimulation of renin
secretion [112]. Further dissection of the mechanism was
obtained by data showing that the integral position of JG
cells within the vascular wall is an absolute requirement for
pressure sensitivity of renin release [130]. Mice deficient in
the gap junction protein connexin 40 (Cx40−/−) dis-
played an unexpected phenotype where JG cells were
ectopically localized, dissociated from the vascular wall
[67]. Disruption of Cx40 led to significantly elevated
blood pressure, loss of inhibition of renin release by
ANG II, and perfusion pressure [130]. This observation
has clinical implications since the human juxtaglomeru-
lar apparatus displays a similar connexin expression as
rodents [62], and a human mutation in Cx40, associated
with hypertension, yields hypertension and ectopic JG
cells when introduced in the mouse [73]. Loss of JG
cell “homing” is associated with hypertension and com-
plete absence of pressure sensitivity of renin secretion.
Together, the data show that pressure-mediated suppres-
sion of renin release depends on adenosine, on extracel-
lular calcium, and on Cx40-dependent proper vascular
localization of JG cells.
32
Renal sympathetic nerves—catecholamines
Renin mRNA abundance and renin release are tonically
stimulated through a β-adrenoceptor mechanism indepen-
dent of tubular function or glomerular filtration rate. Renin
release and renin mRNA abundance are decreased by renal
denervation [46], while α1-adrenoceptor agonists have the
opposite effect in vivo [45]. In addition to pharmacological
data, gene-targeted mice have further supported the notion
that β1-adrenoceptors are necessary for catecholamine-
mediated stimulation of renin secretion in vivo and that
β1-agonist-mediated renin release depends on G protein
Gsα and on adenylyl cyclase types 5 and 6 (Fig. 1) [1,
17]. In vitro, β1-adrenoceptor occupation initiates cAMP
formation in JG cells and increases renin release and renin
mRNA level [18, 24] and cell membrane capacitance, doc-
umenting exocytosis [30]. At present, there are no system-
atic publications documenting the effect of catheter-based
radiofrequency ablation of renal sympathetic nerves in
patients with treatment-resistant hypertension on plasma
concentration of renin–angiotensin system components. In
one minor Dutch study, plasma renin activity did not
change, while plasma aldosterone fell [129]. New data from
many ongoing trials will provide this information in the near
future.
Macula densa and tubular control of renin secretion
It has been established that whole body sodium chloride
content exerts a negative feedback on renin secretion
through the macula densa mechanism [72, 119, 128]. As
defined by Schnermann [98], a true mediator of responses in
the JG apparatus (JGA) must be able to exert a graded effect
on renin secretion, and this effect must be initiated by NaCl
transport-dependent signals which stimulate or inhibit the
local concentration of the mediator. Improper positioning of
JG cells as observed in Cx40−/− mice leads to impaired
stimulation by furosemide [67]. The proper localization of
JG cells is a prerequisite for the macula densa signal. A
close contact between macula densa cells and JG cells is
necessary. A diffusible mediator appears unable to reach
“displaced” JG cells although still in the glomerular tuft
area. Especially, three paracrine mediator candidates have
been in focus: adenosine, NO, and prostaglandins. Macula
densa associated COX-2 mRNA, and protein is regulated
concordantly with renin under most conditions tested (e.g.,
[42, 49, 137]). The sensed variable appears to be the apical
concentration of chloride that correlates inversely to COX-2
expression in cultured TAL/macula densa cells through a
p38 protein kinase-dependent pathway [19, 136]. In isolated
JG apparatus, a low luminal NaCl concentration leads to
release of PGE2 [90]. PGE2 and prostacyclin (PGI2) stimu-
late renin secretion through EP2/EP4/IP receptors on
Pflugers Arch - Eur J Physiol (2013) 465:25–37
juxtaglomerular cells [35, 51, 111]. Mice with targeted
deletion of PGE2-EP4 receptors display low sensitivity of
renin secretion to chronic furosemide [83] and in isolated
perfused kidneys from EP2 and EP4−/− mice, PGE2-mediat-
ed renin secretion is attenuated [111]. In the isolated per-
fused juxtaglomerular apparatus, stimulation of renin
secretion by low luminal NaCl concentration was abolished
by COX-2 blockers [126]. At more integrated levels, a full
dependence of renin secretion on intact COX-2 function has
been difficult to establish. COX-2−/− mice display constitu-
tively low renin expression and exhibit impaired stimulation
of renin by low NaCl intake [135]. Furosemide infusion
yielded a smaller increase in plasma renin in COX-2−/−
compared to wild type [53]. However, combined stimulation
by low salt and AT1 inhibition in COX-2−/− greatly en-
hanced renin stores and markedly increased the release of
renin in response to furosemide [53]. Together, there is good
evidence that COX-2-PGE2 through EP2/EP4 receptors sup-
ports renin release and renin mRNA in conditions with
attenuated transepithelial transport of NaCl in the cortical
loop of Henle. At integrated levels, the findings indicate that
COX-2 rather controls the pool size of renin, perhaps
through effects on the number of JG cells [135], while the
effect on renin secretion is redundant and not mandatory.
Adenosine A1 analogues inhibit renin release from the
isolated JGA when applied to the abluminal side of the
preparation [132] and cause depolarization and calcium
increases in JG cells [69]. Adenosine inhibits renin release
both in vivo and in vitro [48, 59, 118]. In general, vasocon-
strictor A1 receptors are activated by nanomolar concentra-
tions of adenosine, but very high concentrations of
adenosine are necessary to inhibit low NaCl-stimulated re-
nin release from the isolated, perfused juxtaglomerular ap-
paratus [48, 59, 118]. On the other hand, mice with targeted
deletion of adenosine A1 receptors display elevated basal
renin plasma concentration [7] and impaired suppression of
renin by high salt intake [54, 95] and by high perfusion
pressure [112], while stimulation by furosemide was intact
[112, 113]. Current data support a significant role for aden-
osine in both the TGF response [99] and in suppression of
renin secretion in response to high NaCl and elevated per-
fusion pressure, whereas, stimulation of renin by low trans-
epithelial NaCl transport across the loop of Henle is not
dependent on adenosine.
Nitric oxide
Macula densa cells selectively express neuronal nitric oxide
synthase or NOS-I [80]. NOS-I expression in the macula
densa correlates to renin expression (e.g., [110]). NO syn-
thase inhibition reduces renin secretion and renin mRNA
level under acute [102] or chronic [107] low renal perfusion
pressure, under a low dietary NaCl intake [109], under
Pflugers Arch - Eur J Physiol (2013) 465:25–37
furosemide treatment [105], and under ANG II receptor
blockade [106]. Targeted deletion of NOS-I led to lower
plasma renin concentration and lower secretion rates from
isolated perfused kidneys and attenuated furosemide-
stimulated renin secretion [16]. The stimulation of renin
secretion induced by a low luminal NaCl concentration
was abolished in the presence of an NO synthesis inhibitor
[43]. NO is of significance for a low NaCl-induced renin
release at the level of the juxtaglomerular apparatus. NO is
permissive for renin secretion, in particular, during low
NaCl conditions and likely through inhibition of PDE3
(Fig. 1). PDE3 inhibitor administration to nNOS−/− mice
restored plasma renin concentration from low to normal
independent of blood pressure [95].
ANG II
ANG II AT1 receptors are present on JG cells, and ANG II
exposure elicits an increase in the intracellular concentration
of calcium and membrane depolarization, which display
rapid desensitization in JG cells [10, 63, 64]. ANG II is
thought to exert direct negative feedback on renin secretion
by the so-called “short loop feedback inhibition.” Renin
secretion from isolated perfused kidneys in the absence of
nerve input and variation in perfusion pressure is highly
sensitive to ANG II infusion (e.g., [100]). In vitro, the
inhibition of renin release has been difficult to establish
clearly with some exceptions [38]. Despite the presence of
AT1 receptors, recruitment was observed in JG cells in
chimeric mice bearing both AT1+/+ and AT1−/− JG cells,
thus questioning the direct feedback of ANG II on JG cells
[76]. Cross-transplanted mice with AT1A receptor deficien-
cy only in the kidney exhibit a small, but significant lower
blood pressure, while renin expression was not elevated
compared to the AT1A intact mice [23]. This observation
questions the existence of a short-loop feedback, although
plasma renin concentration was not measured. Moreover, in
mice with complete AT1A deletion, renin expression was
elevated, and this corresponded with a lower blood pressure.
This indicates that blood pressure is more important to
determine the renin response after impaired ANG II signal-
ing than short-loop ANG II feedback on renin. Salt-
loading mice with kidney-only AT1 receptor deletion
raised blood pressure and lowered renin expression con-
firming this notion [23]. Quantification of JG cell re-
cruitment in salt-depleted AT1−/− and wild type shows
that recruitment, as well as renin expression, was atten-
uated by increasing blood pressure through, e.g., phen-
ylephrine or L-NAME infusion [74]. Taken together, the
current findings indicate that although functional ANG
II receptors are associated with JG cells, the effect of
ANG II on renin to a significant extent is mediated by blood
pressure.
33
Acknowledgments The authors wish to thank Annette K. Rasmussen
Mette Fredenslund, Lis Teusch, Gitte Dybmose, and Inge Andersen for
their skillful technical assistance. Work in the authors’ lab has been
supported by grants from Carlsbergfondet, the Danish Research Council
for Health and Disease, the Strategic Research Council, the Danish Heart
Foundation, the NOVO Nordisk Foundation, the Lundbeck Foundation,
AP Moller Foundation, Helen and Ejnar Bjørnows Foundation, the
Foundation for the Promotion of Medical Science, and the Foundation
of 17.12.1981.
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