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Lack of Connexin 40 Causes Displacement of Renin-
Producing Cells from Afferent Arterioles to the
Extraglomerular Mesangium
Lisa Kurtz,* Frank Schweda,* Cor de Wit,
†
Wilhelm Kriz,
‡
Ralph Witzgall,*
Richard Warth,* Alexander Sauter,* Armin Kurtz,* and Charlotte Wagner*
*Institut fu¨r Physiologie and Anatomie, Universita¨t Regensburg, Regensburg,
†
Institut fu¨r Physiologie, Universita¨t
Lu¨beck, Lu¨beck, and
‡
Institut fu¨r Anatomie und Embryologie, Universita¨t Heidelberg, Heidelberg, Germany
In the adult kidney, renin-producing cells are typically located in the walls of afferent arterioles at the transition into the
glomerular capillary network. The mechanisms that are responsible for restricting renin expression to the juxtaglomerular
position are largely unknown. This study showed that in mice that lack connexin 40 (Cx40), the predominant connexin of
renin-producing cells, renin-positive cells are absent in the vessel walls and instead are found in cells of the extraglomerular
mesangium, glomerular tuft, and periglomerular interstitium. Blocking macula densa transport function by acute adminis-
tration of loop diuretics strongly enhances renin secretion in vivo and in isolated perfused kidneys of wild-type mice. This
effect of loop diuretics is markedly attenuated in vivo and even blunted in vitro in Cx40-deficient mice. Even after prolonged
stimulation of renin secretion by severe sodium depletion, renin expression is not seen in juxtaglomerular cells or in cells of
more proximal parts of the arterial vessel wall as occurs normally. Instead, renin remains restricted to the extra-/periglomerular
interstitium in Cx40-deficient mice. In contrast to the striking displacement of renin-expressing cells in the adult kidney, renin
expression in the vessels of the developing kidney was found to be normal. This is the first evidence to indicate that cell-to-cell
communication via gap junctions is essential for the correct juxtaglomerular positioning and recruitment of renin-producing
cells. Moreover, these findings support the notion that gap junctions are relevant for the macula densa signaling to
renin-producing cells.
J Am Soc Nephrol 18: 1103–1111, 2007. doi: 10.1681/ASN.2006090953
T
he aspartyl-protease renin is the regulating key enzyme
of the renin-angiotensin-aldosterone system, which
controls BP and extracellular volume. Renin is predom-
inantly produced by the kidneys. Renin-producing cells of the
kidneys show a high degree of plasticity. The cells are com-
monly considered to be a special subset of transformed vascu-
lar smooth muscle cells (VSMC) that are related to myofibro-
blasts or pericytes (1). During angiogenesis and vasculogenesis
of the kidneys, renin-producing cells cover most of the arterial
vascular tree (1– 6). During vessel maturation, renin expression
in proximal parts of the arteriole is progressively silenced so
that in the adult kidney, it is condensed in juxtaglomerular cells
of the ultimate part of the afferent arterioles (1). In this segment
of the arteriole, renin-producing cells largely replace the typical
VSMC. Because of the high number of renin storage vesicles,
the cells achieve a cobble-stone like “epithelioid” appearance.
Chronic stimulation of the renin-angiotensin-aldosterone sys-
tem by extracellular volume depletion or increased sympathetic
nerve activity is associated with re-emergence of renin expres-
sion in cells in the walls of larger renal arteries in a pattern that
is not fully predictable (7–12). The cellular mechanisms that are
responsible for initiating or terminating renin expression dur-
ing vasculogenesis, for directing renin expression to the glo-
merular vascular pole in the adult kidney, and for restarting
renin expression in some but not all cells of larger vessels are
not yet understood. Although local humoral as well as biome-
chanical factors have been considered in this context, no clear
concept has emerged.
In this study, we considered the role of cell-to-cell commu-
nication as a novel determinant of focal renal renin expression.
Previous work established that cell-to-cell communication
through gap junctions is important for the positioning and
differentiation of resident cells (13–19). Within the juxtaglomer-
ular apparatus (JGA), renin-producing cells not only form nu-
merous gap junctions among each other but also are connected
to extraglomerular cells and to endothelial cells of the afferent
arteriole (20–22). The gap junctions of renin-producing cells are
likely formed by connexin 40 (Cx40), which is expressed with
high density in these cells (23–26). The prominent expression of
Cx40 in the JGA is conspicuous, because with the exception of
the electrical conduction system of the heart, Cx40 is almost
exclusively expressed in the endothelium, where it contributes
to propagation of vasodilation (27–31). In the (juxta)glomerular
region, however, Cx40 is expressed by both endothelial and
nonendothelial cells such as renin-producing and intra- and
Received September 3, 2006. Accepted January 18, 2007.
Published online ahead of print. Publication date available at www.jasn.org.
Address correspondence to: Dr. Armin Kurtz, Physiologisches Institut der Uni-
versita¨t Regensburg, D-93040 Regensburg, Germany. Phone: ⫹49-0-941-9432980;
Fax: ⫹49-0-941-9434315; E-mail: armin.kurtz@vkl.uni-regensburg.de
Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1804-1103
extraglomerular mesangial cells (23–26). Other typical endothe-
lial connexins, such as Cx43, are found in larger kidney vessels
but not in the juxtaglomerular endothelial and renin-producing
cells (23–26). In this study, we used mice that lacked Cx40 to
explore the possibility that gap junctional coupling may be
required for the differentiation and positioning of renin-pro-
ducing cells during renal angiogenesis and vasculogenesis, for
the condensation of renin to the JGA of the adult kidney, and
for the recruitment of renin-expressing cells during prolonged
challenges. Moreover, it was of interest for us to assess the role
of Cx40 for the signaling of macula densa cells to renin-pro-
ducing cells, a process that is considered to involve gap junc-
tional function (32).
Materials and Methods
All animal experiments were conducted according to the National
Institutes of Health guidelines for the care and use of animals in
research. Kidneys were sampled from eight 12- to 20-wk-old male
homozygous Cx40⫺/⫺ mice (33), from three fetuses (days 17 to 19),
and from three pups (postnatal day 1). Age-matched wild-type (wt)
mice served as controls. In addition, five adult male Cx40⫹/⫺ mice
were examined. Furthermore, five adult male mice of the Cx40⫹/⫹
and Cx40⫺/⫺ strains were pretreated with a low-salt diet (0.02%
wt/wt) for 1 wk. In addition, these mice received the angiotensin-
converting enzyme inhibitor (ACE) enalapril (10 mg/kg) via the drink-
ing water for the last3dofthedietary treatment. The genetic back-
ground of Cx40-deficient and wt mice was considered identical because
Cx40-deficient mice were backcrossed for seven generations on a
C57Bl/6 background. The genotype of the mice was verified by PCR as
described previously (34).
For assessment of the macula densa control of renin secretion in vivo,
Cx40⫺/⫺ and Cx40⫹/⫹ mice of either gender (25 to 30 g body wt)
were used (Cx40⫹/⫹ seven male and seven female mice; Cx40⫺/⫺
eight male and eight female mice). For determination of plasma renin
concentration (PRC), blood samples were taken from the tail vein. Ten
days after baseline blood collection, all mice received a single injection
of furosemide (40 mg/kg body wt; Dimazon, Intervet, Germany) and a
blood sample was taken 60 min thereafter from the tail vein.
Immunohistochemistry for Renin and
␣
-Smooth Muscle Actin
The expression of renin and
␣
-smooth muscle actin (
␣
-SMA) was
localized by immunohistochemistry. In brief, kidneys were fixed in
methyl-Carnoy solution (60% methanol, 30% chloroform, and 10% gla-
cial acetic acid) as described previously (35). Immunolabeling was
performed on 5-
m paraffin sections. After blocking with 10% horse
serum and 1% BSA in PBS, sections were incubated with anti-renin or
anti–
␣
-SMA antibodies (Beckman Coulter, Immunotech, Marseilles,
France) overnight at 4°C, followed by incubation with a fluorescence
secondary antibody.
Confocal Microscopy
Sections were analyzed with a confocal microscope (LSM 510; Zeiss,
Go¨ttingen, Germany) using sequential scanning (Plan Apochromat
63⫻/1.4 oil objective, excitation at 488 and 543 nm, emission at 505 to
530 and 560 to 615 nm, respectively).
Bromodeoxyuridine Incorporation
Bromodeoxyuridine (BrdU; Roche, Mannheim, Germany) incorpora-
tion into nuclei was determined as described previously (36). In brief,
BrdU dissolved in sterile isotonic saline (10 mg/ml) was injected twice
daily (0.1 mg BrdU/g body wt per d, intraperitoneally) starting on the
day before the enalapril treatment period. After a total of 7 d, the mice
were anesthetized with an intraperitoneal injection of 100 mg/kg 5-eth-
yl-5-(1-methylbutyl)-2-thiobarbituric acid and 80 mg/kg ketamine-HCl
and fixed by vascular perfusion with 4% paraformaldehyde. After the
removal of kidneys and intestine and the embedding in paraffin, 5-
m
sections were incubated in 4 M HCl for 30 min, neutralized by 0.1 M
sodium borate (pH 8.5), and finally incubated in 0.1% trypsin in PBS.
After blocking with 2% BSA and 0.1% TritonX-100 in PBS, sections were
incubated with anti-BrdU antibody (1:1000) overnight, followed by
incubation with a fluorescence secondary antibody.
Isolated Perfused Mouse Kidney
Male Cx40⫺/⫺ and age-matched Cx40⫹/⫹ mice were used as kid-
ney donors. The isolated perfused mouse kidney model was described
in detail previously (37). Briefly, the mice were anesthetized with an
intraperitoneal injection of 12 mg/kg xylazine (Rompun; Bayer, Wup-
pertal, Germany) and 80 mg/kg ketamine-HCl (Curamed, Karlsruhe,
Germany); the abdominal aorta was cannulated; and the right kidney
was excised, placed in a thermostated moistening chamber, and per-
fused at constant pressure (90 mmHg). Finally, the renal vein was
cannulated and the venous effluent was collected for determination of
renin activity and venous blood flow.
The basic perfusion medium consisted of a modified Krebs-Henseleit
solution supplemented with 6 g/100 ml BSA and with freshly washed
human red blood cells (10% hematocrit). Stock solutions of isoproter-
enol were dissolved in freshly prepared perfusate; stock solution of
bumetanide was made in DMSO. All drugs were infused into the
arterial limb of the perfusion circuit.
For the determination of renin secretion rates, three samples of the
venous effluent were taken in intervals of 2 min during each experi-
mental period. Renin activity in the venous effluent was determined by
RIA (Byk & DiaSorin Diagnostics, Dietzenbach, Germany) as described
previously. Renin secretion rates were calculated as the product of the
renin activity and the venous flow rate (ml/min ⫻ g kidney weight).
Determination of PRC
For determination of PRC, the blood samples that were taken from
the tail vein were centrifuged and the plasma was incubated for 1.5 h
at 37°C with plasma from bilaterally nephrectomized male rats as renin
substrate. The generated angiotensin I (ng/ml per h) was determined
by RIA (Byk & DiaSorin Diagnostics).
Electron Microscopy
Kidneys were perfusion-fixed and embedded in Epon according to
standard procedures. One-micrometer sections of several blocks in-
cluding a series of 200 1-
m sections were cut with a diamond knife,
stained with methylene blue (38), and studied by light microscopy.
Ultrathin sections were prepared from selected areas and studied by a
Philips 307 transmission electron microscope (Eindhoven, The Nether-
lands).
Three-Dimensional Reconstruction
Serial sections of kidney specimen were fixed and stained for renin
and for
␣
-SMA as described in the previous section. A three-dimen-
sional (3-D) reconstruction of renin immunoreactivity and of
␣
-SMA
immunoreactivity was performed using the Amira 3.1 visualization
program (Mercury Systems, Merignac, France).
1104 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1103–1111, 2007
Results
In adult kidneys of Cx40⫹/⫹ mice, renin-expressing cells
were restricted to the vascular poles of the glomeruli. Renin-
producing cells were integrated into the regular wall of the
afferent arterioles in continuation of the vessel lined by VSMC
(Figure 1, A and B). The latter cells were identified by the
expression of
␣
-SMA (Figure 1, A and B). Renin-producing cells
at the very end of the afferent arterioles showed the typical
epithelioid appearance (juxtaglomerular epithelioid cells) and
expressed no
␣
-SMA. Mixed-phenotype cells that expressed
both renin and
␣
-SMA were found to be located between the
typical SMC and juxtaglomerular epithelioid cells (Figure 1, A
and B). The distribution of renin-expressing cells in Cx40⫹/⫺
kidneys was very similar to that in kidneys of wt mice (data not
shown).
In contrast, the number of renin-expressing cells in adult
kidneys of Cx40-deficient mice was seemingly increased when
compared with wt controls. The number of renin-producing
cells in individual periglomerular regions of Cx40⫺/⫺ kidneys
was variable, ranging from 1 to 20 cells per glomerular region.
Renin-positive cells in Cx40⫺/⫺ kidneys were restricted to the
juxtaglomerular/periglomerular interstitium (Figure 1, C and
D). As confirmed by confocal microscopy, renin-producing
cells in the juxtaglomerular region were not integrated into the
wall of afferent arterioles but instead surrounded the vessel
wall that consisted of endothelial cells and
␣
-SMA–positive
VSMC (Figure 1, E and F). Moreover, renin-producing cells
extended into the region of the extraglomerular mesangium
(EGM) and into the periglomerular interstitial space between
tubules and around glomeruli. The cells did not show the
classic epithelioid appearance but rather appeared mesen-
chyme-like and irregularly shaped. The cytosol of these cells
showed renin immunoreactive granulation. All renin-positive
cells stained negative for
␣
-SMA. No renin-producing cells
were associated with larger kidney vessels (Figure 1, C and D).
Electron microscopic analysis confirmed that walls of affer-
ent arterioles were free of granular cells (Figure 2C). Further-
more, VSMC were found inside the glomerular stalk of
Cx40⫺/⫺ kidneys, whereas they normally terminate at the
entrance into the JGA. VSMC were also observed in the intra-
glomerular segment of the efferent arteriole that is normally
surrounded by mesangial cells and mesangial cell processes. In
contrast to wt kidneys, the majority of extraglomerular mesan-
gial cells in Cx40⫺/⫺ mice contained granules as they are
normally seen in renin-producing cells of the afferent arteriole
(Figure 2, A through D). For example, tracing of an entire
glomerulus in a series of 1-
m sections revealed no cells in the
EGM without granules (Figure 2B). In addition, granular cells
were found in the glomerular stalk to a variable extent. Occa-
sionally, the intraglomerular segment of the efferent arteriole
deep within the glomerular tuft also appeared granulated.
In other sites, EGM cells that looked fairly normal with
elongated processes and without any granules were found
(Figure 2E). The interstitial spaces between these cells seemed
wider and were filled with a translucent amorphous matrix.
Gap junctions were not encountered. Within the intraglomeru-
lar mesangium, foci of mesangiolysis that may be the result of
mesangial cell proliferation and matrix production, leading to
local mesangial expansions, were noted. The resulting narrow-
ing of associated capillaries and their incorporation into the
proliferating mesangium may cause obstruction and eventual
degeneration.
In Cx40⫹/⫹ mice that received a low-sodium (0.02% wt/wt)
diet and the ACEI ramipril, a clear increase of renin-expressing
cells appeared in the walls of the preglomerular vessels (Figure
3, A and B). Cells that expressed both
␣
-SMA and renin could
be seen with increased frequency. In contrast, no expression of
renin was found in the walls of the arterioles or larger vessels
of Cx40⫺/⫺ kidneys (Figure 3, C and D). Instead, the number
of renin-expressing periglomerular cells seemed to be in-
Figure 1. Immunohistochemistry for renin (green) and
␣
-smooth muscle actin (
␣
-SMA; red) on kidney sections of
wild-type (wt; A and B) and of connexin 40 deficient
(Cx40⫺/⫺; C and D) mice. In wt mice, renin immunoreactivity
was found in the walls of afferent arterioles close to the glo-
meruli (dotted, G). Renin-positive cells had a regular form,
appeared thicker, and were negative for
␣
-SMA at the very end
of the afferent arterioles. In Cx40⫺/⫺ kidneys, the number of
renin-positive cells in the juxtaglomerular region was clearly
increased. The cells had a more irregular shape, and they were
not integrated into the walls of the afferent arterioles. Impres-
sion was further corroborated by a confocal analysis (E and F)
of kidney sections from Cx40⫺/⫺ mice, showing that renin-
positive cells were separated from endothelial cells by a layer of
␣
-SMA–positive smooth muscle cells. *, Macula densa; arrow-
heads, endothelial cells. Bars ⫽ 20
m.
J Am Soc Nephrol 18: 1103–1111, 2007 Connexin 40 and Displacement of Renin-Producing Cells 1105
creased. Cx40⫹/⫺ behaved like Cx40⫹/⫹ kidneys (data not
shown).
When the number of renin-expressing cells in the periglo-
merular region was particularly high, a clear expansion of the
periglomerular cell mass could be readily detected by light
microscopy (Figure 3, E and F). Otherwise the histology of the
kidneys was normal, except for the appearance of sclerotic
glomeruli. To determine whether expansion of periglomerular
cell mass was associated with proliferation of interstitial cells,
we determined the number of cells that underwent an S phase
during treatment with low salt and the ACEI. Using the BrdU
incorporation method, we found single BrdU incorporating
nuclei in proximal tubules but no BrdU incorporation into
nuclei of renin-immunoreactive cells, neither in wt nor in
Cx40⫺/⫺ kidneys (data not shown).
Because the extent of periglomerular renin expression was
difficult to estimate from single histologic sections, we per-
formed a 3-D reconstruction of 80 consecutive 5-
m sections
that stained for renin and for
␣
-SMA. With the use of this
technique, no obvious differences between preglomerular ves-
sel trees of wt and Cx40⫺/⫺ kidneys were noted (Figure 4, A
and B). The number of glomeruli within this defined kidney
volume was similar between wt and Cx40⫺/⫺ kidneys. In
low-salt/ACEI-treated wt mice, renin expression was exclu-
sively associated with the walls of the distal parts of the afferent
arterioles, where the renin-producing cells formed cuff-like
Figure 2. (A) High-resolution light microscopy longitudinal sec-
tion (1
m) through the juxtaglomerular apparatus (JGA) and
glomerulus. Granular cells are abundant and are encountered at
abnormal sites in the extraglomerular mesangium (EGM; *) and
the glomerular stalk (arrow). (B) Oblique section through the JGA
showing the abundance of granular cells in the EGM and as
disseminated cells everywhere in the surroundings of the JGA
(arrows). (C) Transmission electron microscopy longitudinal sec-
tions through the JGA. Granular cells (G) are seen within the
EGM; a vascular smooth muscle cell (SM) extends into the glo-
merular stalk. (D) Transmission electron microscopy longitudinal
sections through the JGA. Beneath the macula densa (MD), a
group of EGM cells that contain abundant granules of variable
electron density are seen (arrows); also protogranules (arrow-
heads) are regularly encountered. (E) Transmission electron
microscopy longitudinal sections through the JGA. Beneath the
macula densa are a group of EGM cells and processes that look
fairly normal. Gap junctions have not been encountered be-
tween the EGM cells. AA, afferent arteriole; EA, efferent arte-
riole. Magnifications: ⫻500 in A; ⫻625 in B; ⫻1800 in C; ⫻5700
in D; ⫻4500 in E.
Figure 3. (A through D) Immunohistochemistry for renin
(green) and
␣
-SMA (red) on kidney sections of wt (A and B)
and of Cx40⫺/⫺ (C and D) mice treated with a combination of
low salt and the angiotensin-converting enzyme inhibitor
(ACEI) ramipril. In the kidneys of wt mice, renin expression
was increased in the walls of the preglomerular vessels. Co-
localization of renin and of
␣
-SMA (yellow) was increased. In
kidneys from Cx40⫺/⫺ mice, again no renin immunoreactivity
became visible in the vessel walls. Instead, renin expression
spread out into the periglomerular-peritubular space. (E and F)
Consecutive kidney sections of Cx40⫺/⫺ mice treated with
low salt and the ACEI stained with renin/
␣
-SMA (E) and with
hematoxylin-eosin (F). Bars ⫽ 20
m.
1106 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1103–1111, 2007
structures at the vascular pole of virtually all glomeruli. In
Cx40⫺/⫺ kidneys, no cuff-like structures were observed. In-
stead, 30% of glomeruli showed a prominent periglomerular
spreading (Figure 4B). In the remaining glomeruli, the number
of renin-expressing cells was markedly lower, but again these
cells were not integrated into the walls of the afferent arterioles.
In view of the aberrant position of renin-producing cells in
the kidneys of adult Cx40⫺/⫺ mice, it was of interest to study
the localization of renin-producing cells during the develop-
ment of the kidneys. Therefore, we performed a 3-D analysis of
the vascular tree and of renin-producing cells of kidneys at day
18 after conception. As shown in Figure 4, C and D, the differ-
ent arteries and arterioles could clearly be identified in embry-
onic kidneys of wt and Cx40⫺/⫺ mice. There were no obvious
differences in the vascular morphology in the two genotypes.
Similarly, the distribution of renin-producing cells in these
fetal kidneys was very similar between the genotypes. The
renin-producing cells were associated with the walls of the
larger arteries. Most notable, renin expression in Cx40⫺/⫺
kidneys was restricted to the vessel wall and did not appear in
the interstitium (Figure 4, C and D). The first clearly recogniz-
able divergence in the localization of renin-producing cells
between the two genotypes appeared with the development of
renin expression in the glomerular regions. Figure 5, B through
D, shows that in the kidneys of 1-d-old Cx40⫺/⫺ pups, renin-
expressing cells are still integrated into the walls of afferent
arterioles, although they start to spread out at the vascular
poles. In wt kidneys of the same age, renin expression remained
restricted to the vessel wall of the preglomerular arteries (Fig-
ure 5A).
Considering these apparent changes of the architecture of the
JGA raises the question about functional changes. It was of
particular interest for us to see whether the normal signaling of
the macula densa cells to renin-producing cells is altered in the
absence of Cx40. To study the macula densa control of renin
secretion, we used a classical maneuver, the acute administra-
tion of loop diuretics, which blocks salt transport in the thick
ascending loop of Henle including the macula densa segment.
In Cx40⫹/⫹ mice, application of furosemide led to a 15-fold
increase of PRC (Figure 6A). Similarly, also in isolated perfused
kidneys of Cx40⫹/⫹ mice, bumetanide caused a strong en-
hancement of renin secretion (Figure 6B). In Cx40⫺/⫺ mice,
basal PRC were sixfold elevated when compared with
Cx40⫹/⫹ mice (Figure 6A). Administration of furosemide in-
creased PRC values only two-fold in Cx40⫺/⫺ mice (Figure
7A). Renin secretion from isolated perfused kidneys of
Cx40⫺/⫺ mice responded well to isoproterenol, whereas the
response to bumetanide was absent (Figure 6B).
Figure 4. (A and B) Three-dimensional (3-D) reconstruction of
serial kidney sections stained for renin/
␣
-SMA of adult wt (A)
and Cx40⫺/⫺ (B) kidneys. Mice were pretreated with low salt
and ACEI (green, renin; red,
␣
-SMA). (C and D) 3-D reconstruc-
tion of serial kidney sections stained for renin and for
␣
-SMA of
wt (C) and of Cx40⫺/⫺ (D) mice at embryonic day 18 (green,
renin; red,
␣
-SMA). Bars ⫽ 500
m.
Figure 5. Immunohistochemistry for renin (green) and
␣
-SMA
(red) on kidney sections of wt (A) and Cx40⫺/⫺ (B through D)
mice 1 d after birth. Yellow indicates overlap of renin and
␣
-SMA. Bars ⫽ 20
m.
J Am Soc Nephrol 18: 1103–1111, 2007 Connexin 40 and Displacement of Renin-Producing Cells 1107
Discussion
A main finding in the present study is that the absence of
Cx40 is associated with a major disturbance in the location and
identity of renin-expressing cells. Cell-to-cell communication
through Cx40 seems to be required for the maintenance of the
architecture of the JGA and the direction of renin expression to
cells in the walls of afferent arterioles in a typical juxtaglomer-
ular location (Figure 6, A and B). In the absence of Cx40,
renin-expressing cells are found outside the terminal parts of
the afferent arterioles and extending into the juxta- and peri-
glomerular interstitium. Furthermore, stimulation of renin syn-
thesis and secretion does not induce the normal recruitment of
renin-expressing cells in the walls of upstream preglomerular
arteries.
Given that Cx40 is the main gap junctional protein in the JGA
(25–28), especially in the region of renin production (Figure
Figure 6. Effect of loop diuretics on renin secretion in vivo (A) and
from isolated perfused kidneys (B) in Cx40⫹/⫹ and Cx40⫺/⫺
mice. (A) Data are means ⫾ SEM of 14 and 16 Cx40⫹/⫹ and
Cx40⫺/⫺ mice, respectively. Furosemide (40 mg/kg) was in-
jected 10 d after baseline blood collection of each mouse. Plasma
renin concentration values were determined 1 h after injection of
furosemide. #P ⬍ 0.001 versus baseline Cx40⫹/⫹;*P ⬍ 0.05 versus
baseline Cx40⫺/⫺. (B) For isolated perfused kidneys, five kidneys
of each genotype were used. Data are means ⫾ SEM. E,
Cx40⫺/⫺; F, Cx40⫹/⫹. The increases of renin secretion that
were elicited by isoproterenol (iso; 3 nM) were significant (P ⬍
0.05) for both genotypes. The change of renin secretion that was
elicited by bumetanide (bum) was significant for Cx40⫹/⫹ only
(P ⬍ 0.05 versus iso 3 nM alone). The change of renin secretion that
was elicited by iso (10 nM) was significant (P ⬍ 0.05 versus iso 3
nM ⫹ bum 100
M) for both genotypes.
Figure 7. Sketch summarizing the morphology of the JGA in the
presence (A) and absence (B) of Cx40. mdc, macula densa cells;
JGE, juxtaglomerular epithelioid cells; ec, endothelial cells; mc,
mesangial cells; gcec, glomerular capillary endothelial cells;
pod, podocytes; vsmc, vascular smooth muscle cells; Bc, Bow-
man capsule.
1108 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1103–1111, 2007
6A), our observations are in accordance with numerous find-
ings of a central role of gap junctions in the differentiation of a
variety of cells in the cardiovascular, nervous, or reproductive
system (13–19). There is general agreement that gap junctional
communication favors structural and functional differentiation,
whereas loss of gap junctions leads to dedifferentiation (13–19).
There is also agreement that gap junctional communication is
important for the correct formation of endo- and exocrine
glands and for coordinating the responses of individual gland
cells (39).
Our data suggest that the mislocation of renin-producing
cells in Cx40⫺/⫺ kidneys may begin with typical expression of
renin in the juxtaglomerular region. Endothelial cells of the
afferent arterioles, renin-producing cells, and mesangial cells
form a network of cells that are connected via Cx40 gap junc-
tions (23–26) (Figure 6C) and that are derived from the juxta/
periglomerular mesenchyme as a group of pericytes. Notably,
all of these cell types, including the extraglomerular and intra-
glomerular mesangial cells (40,41), have the capability to syn-
thesize renin, although the expression of renin in the adult
mesangium is a very rare event (1,42–44).
Because renin expression in the EGM of Cx40⫺/⫺ kidneys is
abundant, Cx40 gap junctions do not seem to be a general
prerequisite for the expression of renin in the pericytes, a
conclusion that is confirmed by normal fetal development.
Because endothelial cells in the afferent arteriole normally form
Cx40 gap junctions with renin-producing cells but not with
VSMC, one may speculate that the lack of Cx40 gap junctions
leads to a transformation of originally renin-producing peri-
cytes into VSMC. As a consequence, remaining renin-produc-
ing cells are displaced to the periphery of the afferent arterioles,
initiating the process of dispersion of renin expression to cells
outside the afferent arteriolar vessel wall. Our findings suggest
that this proliferation period ends once the formation of the
glomeruli is complete.
One may hypothesize that contact between endothelial cells
and renin-producing pericytes via Cx40 gap junctions could
provide an essential anchor for the juxtaglomerular position of
renin-producing cells. Once a stable contact is established, en-
dothelium-coupled pericytes may send a signal through Cx40
gap junctions to neighboring pericytes to suppress their prolif-
eration and renin secretion. The inherent capability to produce
renin, however, would be preserved. In fact, it is known that
long-lasting stimulation of the renin-angiotensin system can
lead to the recruitment of renin production not only in the
larger vessels but also in the EGM (42–47).
The displacement of renin-producing cells raises the question
of whether the physiologic regulation of the renin system is
affected by the absence of Cx40. It is known that Cx40⫺/⫺
mice are hypertensive (48). Although first evidence suggests
that the hypertension in Cx40⫺/⫺ mice is not primarily renin
dependent (48), our observation of elevated PRC values (Figure
7A) could suggest that an exaggerated renin secretion could
contribute to the hypertension. The enhanced basal renin secre-
tion in Cx40⫺/⫺ mice could result from an interruption of the
inhibitory macula densa signaling to renin-producing cells.
Such an explanation would be supported by our findings that
the stimulatory effect of loop diuretics on renin secretion is
absent in isolated kidneys of Cx40⫺/⫺ mice (Figure 7B) and is
markedly attenuated in vivo (Figure 7A). A possible explanation
for why the renin response to loop diuretics in vivo is not
blunted as in vitro is that additional factors may contribute to
the effects of loop diuretics in vivo, such as inhibition of NKCC1
(49,50) or activation of the sympathetic nervous system, which
may be less relevant in vitro. In fact, our in vitro findings suggest
that Cx40⫺/⫺ kidneys respond more sensitively to

-adreno-
receptor activation than do Cx40⫹/⫹ kidneys (Figure 7B).
Altogether, our data show that the macula densa signaling to
renin-producing cells is markedly deteriorated in the absence of
Cx40⫺/⫺, suggesting an important role of this connexin in
macula densa signaling and supporting the conclusions of a
recent elegant imaging study (32).
For further elucidation of the possible underlying mecha-
nisms of the crucial effect of Cx40 for the position of renin-
producing cells and for the macula densa control function, two
strategies will be used. First, in mice with an endothelium-
specific deletion of Cx40, we would expect an interruption of
the communication between endothelial cells and renin-pro-
ducing cells without affecting the communication between re-
nin-producing cells and mesangial cells. Second, replacement
of Cx40 with other related connexins should provide informa-
tion about the connexin subtype that is causal in determining
the position of renin-producing cells.
Acknowledgments
This work was financially supported by the Sonderforschungsbe-
reich 699, by the Deutche Forschungsgemeinschaft grant Wi 2071/1-1,
and by the “Prof. Dr. Karl und Gerhard Schiller-Stiftung, Frankfurt/
Main.”
We thank Jurgen Schnermann for critical reading of the manuscript
and for helpful comments. The expert technical assistance provided by
Anna Ba´ngui, Katharina Machura, and Hiltraud Hosser is gratefully
acknowledged.
Disclosures
None.
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