Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2007;100:556-563
Published online before print January 25, 2007, doi: 10.1161/01.RES.0000258856.19922.45
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
100/4/556    most recent
01.RES.0000258856.19922.45v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wagner, C.
Right arrow Articles by Schweda, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wagner, C.
Right arrow Articles by Schweda, F.
Related Collections
Right arrow Endothelium/vascular type/nitric oxide
Right arrow Cell signalling/signal transduction
Right arrow Genetically altered mice
(Circulation Research. 2007;100:556.)
© 2007 American Heart Association, Inc.

Cellular Biology

Connexin40 Is Essential for the Pressure Control of Renin Synthesis and Secretion

Charlotte Wagner*, Cor de Wit, Lisa Kurtz, Christian Grünberger, Armin Kurtz, Frank Schweda*

From the Physiologisches Institut der Universität Regensburg (C.W., L.K., C.G., A.K., F.S.), Germany; Physiologisches Institut der Universität Lübeck (C.d.W.), Germany.

Correspondence to Charlotte Wagner, PhD, Physiologisches Institut der Universität Regensburg, D-93040 Regensburg, Germany. E-mail charlotte.schmid{at}vkl.uni-regensburg.de


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Renin secretion and synthesis in renal juxtaglomerular cells are controlled by short feed back loops involving angiotensin II and the intrarenal blood pressure. The operating mechanisms of these negative feed back regulators are widely unknown, except for the fact that both require calcium to exert their inhibitory action. We here show that in the absence of connexin40 (Cx40), which form gap junctions between juxtaglomerular and endothelial cells, the negative control of renin secretion and synthesis by angiotensin II and by intravasal pressure is abrogated, while the regulation by salt intake and ß-adrenergic stimulation is maintained. Renin secretion from Cx40-deficient kidneys or wild-type kidneys treated with the nonselective gap junction blocker 18{alpha}-glycyrrhetinic acid (10 µmol/L) resembles the situation in wild-type kidneys in the absence of extracellular calcium. This disturbed regulation is reflected by an enhanced plasma renin concentration despite an elevated blood pressure in Cx40-deficient mice. These findings indicate that Cx40 connexins and likely intercellular communication via Cx40-dependent gap junctions mediate the calcium-dependent inhibitor effects of angiotensin II and of intrarenal pressure on renin secretion and synthesis. Because Cx40 gap junctions are also formed between renin producing cells and endothelial cells our finding could provide additional information to suggest that the endothelium may be strongly involved in the control of the renin system.


Key Words: gap junctions • connexin40 • renin • angiotensin II • perfusion pressure


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The protease renin, mainly produced by the kidneys, is the rate limiting enzyme determining the activity of the renin-angiotensin-aldosterone system (RAAS), which controls sodium homeostasis and blood pressure in mammals.1,2 Renin synthesis and secretion are regulated by the sodium balance of the organism in the sense of a long negative feedback loop.1,2 They are moreover, regulated by angiotensin II (Ang II) as the product of renin activity and by the intrarenal blood pressure, which form short negative feed back loops for renin secretion.1,2 The mechanisms by which the renal perfusion pressure, which is also referred to as the renal baroreceptor,1,2 or Ang II inhibit synthesis and secretion of renin in juxtaglomerular cells are widely unknown.1 However, the inhibitor effects of both factors are abolished if the extracellular calcium concentration is lowered toward the micromolar range,3,4 suggesting a requirement for calcium which possibly acts as the inhibitory mediator in these 2 signaling pathways. It is known for long that juxtaglomerular cells are strongly coupled among each other as well as to the adjacent endothelial cells via gap junctions.5–7 However, the functional implication of this coupling is still obscure partially because of the lack of substances that specifically interfere with gap junctional communication. Gap junctions are formed by the assembly of connexins (Cx), which belong to a family consisting of at least 20 members.8 More recently, it was demonstrated that connexin40 (Cx40) and Cx43 seem to be the dominating connexins expressed in the kidney. Specifically the renin producing juxtaglomerular cells but not the neighboring smooth muscle cells exhibit a striking expression of Cx40 which forms gap junctions between the renin producing cells themselves as well as between the renin producing cells and the adjacent endothelial cells or the extraglomerular mesangium.9–12 Because Cx40-deficient mice are hypertensive,13 we hypothesized that either Cx40 hemi channels or Cx40-dependent intercellular communication is crucially contributing to the regulation of renin secretion. In fact, a recent study provided first evidence that gap junctional communication may have functional relevance for renin producing cells. Mice in which Cx43, which is found in endothelial cells but not in renin producing juxtaglomerular cells,10–12,14 had been replaced by Cx32 (Cx43ki32 mice) displayed a markedly lower renin expression than their respective wild-type (WT) controls.15 More strikingly, unilateral renal artery stenosis which stimulates renin synthesis and secretion in the hypoperfused kidney and suppresses both parameters in the contralateral intact kidney in WT mice, failed to induce these characteristic changes in Cx43ki32 mice.15 How the replacement of Cx43 by Cx32 can exert such profound changes in the functional behavior of juxtaglomerular cells is yet unknown.

Our interest in the functional role of gap junctions for renin synthesis and secretion focused on Cx40, which is the dominating connexin in juxtaglomerular cells. Therefore, we studied the regulation of renin synthesis and secretion in Cx40 deficient mice, which are known to be hypertensive for unknown reasons. We aimed to prove 2 hypotheses, namely that the hypertension is because of a dysregulation of renin secretion and if so, that the dysregulation of renin is because of a disturbed feed-back control of renin secretion.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animal Experiments
All experiments were conducted in 12 to 20 weeks old homozygous Cx40–/– mice16 and age-matched WT controls. Strain background (C57BL/6) of Cx40-deficient and WT animals were considered identical after Cx40-deficient mice were backcrossed seven times in a C57BL6 background. To determine the gene dose effect of Cx40 the phenotype of Cx40+/– mice was determined in a second step. The genotype of all animals was verified by PCR as described previously.17

All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the local ethics committee.

Male mice were assigned to the following groups: (a) Enalapril treatment: 8 mice of each genotype were given the converting enzyme inhibitor enalapril (10 mg kg–1 d–1) for 5 days in the drinking water. Control mice (n=8 each genotype) received normal tap water; (b) Salt diets: Mice were maintained for 10 days on chow balanced in all respects except for a low (0.02% NaCl, Ssniff) or a high (4% NaCl, Ssniff) sodium content (n=8 for each treatment and genotype); (c) Isoproterenol treatment: Isoproterenol (10 mg kg–1 d–1) was administered continuously via subcutaneously implanted osmotic minipumps (Alzet Corp) for 2 days18; control animals received 0.9% NaCl via osmotic minipumps (n=5 for each genotype and treatment); and (d) Unilateral renal artery stenosis: Animals were anesthetized with sevoflurane, the left kidney was exposed by a small flank incision and the renal artery was dissected from the renal vein and the surrounding tissue. Finally a U-shaped silver clip (0.11-mm-inner-diameter) was placed around the renal arteries of 8 mice of each genotype, whereas no clip was placed in sham operated mice (n=8 each genotype). Animals were euthanized 7 days after clipping.

After the respective treatment periods blood samples (75 µL) for the determination of the plasma renin concentration were taken from the tail vein into hematocrit tubes containing 1 µL 125 mmol/L EDTA to prevent clotting. Thereafter, the mice were deeply anesthetized with sevoflurane and killed by cervical dislocation. The kidneys were removed quickly and frozen in liquid nitrogen. Organs were stored at –80°C before isolation of mRNA or determination of tissue renin activity, as described below.

Blood Pressure Measurements
Measurements of the systolic arterial pressure were performed noninvasively by tail cuff manometry (TSE, Germany). Before the first blood pressure determination the animals were habituated on the experiment procedure by placing them into the holding device on 5 sequent days.

Determination of Renin mRNA by Real-Time PCR
Total RNA was isolated from the frozen kidneys as described by Chomczynski and Sacchi.19 The cDNA was synthesized by MMLV reverse transcriptase (Superscript, Invitrogen). For quantification of renin mRNA and Cx43 mRNA expression real time RT-PCR was performed using a Light Cycler Instrument (Roche Diagnostics Corp) and the QuantiTect SYBR Green PCR kit (Quiagen) and ß-actin as a control. To verify the accuracy of the amplicon a melting curve analysis was done after amplification and PCR products were analyzed on an ethidium bromide-stained agarose gel. For amplification of mouse renin and ß-actin cDNAs the following primers were used: renin: 5'-atg aag ggg gtg tct gtg ggg-3' (sense), 5'atg cgg gga ggg tgg gca cct-3' (antisense); ß-actin: 5'- cgg gat ccc cgc cct agg cac cag ggt g-3' (sense), 5' - gga att agg ctg ggg tgt tga agg tct caa a-3'(antisense).

Determination of Plasma Renin Concentration
For determination of plasma renin concentration the blood samples taken from the tail vein were centrifuged and the plasma was incubated for 1.5 hour at 37°C with plasma from bilaterally nephrectomized male rats as renin substrate. The generated AngI [ng/mLxh] was determined by radioimmunoassay (Byk & DiaSorin Diagnostics, Germany).

Determination of Renal Renin Content
Renal renin content was determined as described previously.20 In brief, frozen kidney halves were homogenized and centrifuged. The supernatant was refrozen and thawed three times to activate renin. Renin activity was determined by radioimmunoassay using saturating concentrations of renin substrate.20 Protein was determined with the Bradford-method (BioRad).

Isolated Perfused Mouse Kidney
Homozygous Cx40 deficient mice and age-matched WT mice of either sex with a C57BL/6 genetic background were used as kidney donors. The isolated perfused mouse kidney model has been described in detail previously.20 Briefly, the animals were anesthetized with an intraperitoneal injection of 12 mg/kg xylazine (RompunR, Bayer, Germany) and 80 mg/kg ketamine-HCl (Curamed, Germany), the abdominal aorta was cannulated, the right kidney was excised, placed in a thermostated moistening chamber and perfused at constant pressure (90 mm Hg). Using an electronic feedback control, perfusion pressure could be changed and held constant in a pressure range between 40 and 140mmHg. 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 bovine serum albumin and with freshly washed human red blood cells (a 10% hematocrit).

Stock solutions of Ang II or isoproterenol were dissolved in freshly prepared perfusate; stock solutions of 18{alpha}-glycyrrhetinic acid or glycyrrhizic acid (GZA) were dissolved in DMSO. All drugs were infused into the arterial limb of the perfusion circuit. For lowering the extracellular calcium concentration into the submicromolar range we added the calcium-chelator ethylene glycol-bis (aminoetyhl ether) tetraacetic acid (EGTA) 3,12mMol/L to the perfusate. The experiments with GZA were performed in separate experiments in a second step.

For the determination of renin secretion rates three samples of the venous effluent were taken in intervals of 2 minutes during each experimental period. Renin activity in the venous effluent was determined by radioimmunoassay (Byk & DiaSorin Diagnostics, Germany) as described previously.20 Renin secretion rates were calculated as the product of the renin activity and the venous flow rate [ml/min*g kidney weight].

Isolation of JG cells and Primary Cell Culture
Mouse JG cells were isolated as described.21 Briefly, cells from 4 mouse kidney homogenates were separated by Percoll gradient centrifugation (n=4 preparations), and the cellular layer (density 1.07 g/mL) with the highest specific renin activity was washed and resuspended in 4 mL RPMI-1640 (Biochrom) containing 0.66 U/mL insulin, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2% FCS. Cells were seeded in 100-µL aliquots in 96-well cell culture plates and were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. After 20 hours of primary culture, the medium was removed, the cultures were washed once with 100 µL RPMI-1640 medium containing 2% FCS and finally 100 µL of prewarmed culture medium with the chemicals to be tested (forskolin 5 µmol/L; Ang II 1µmol/L; thapsigargin 1µmol/L) were added and incubation was continued for 4 hour. Thereafter, the supernatants were removed and renin activity was determined by the ability of the samples to generate Ang I from the plasma of bilaterally nephrectomized rats. Ang I was measured by radioimmunoassay (Byk & DiaSorin Diagnostics, Germany).

Immunohistochemistry for Renin, Cx40, Cx43, and {alpha} Smooth Muscle Actin
The expression of renin, Cx40, and Cx43 protein were localized by immunohistochemistry.

In brief, kidneys were fixed in methyl-Carnoy solution (60% methanol, 30% chloroform, 10% glacial acetic acid) as described previously.22 Immunolabeling was performed on 8 µm frozen sections. After blocking with 10% horse serum, 1% BSA in PBS, sections were incubated with anti-renin, anti Cx40 (Santa Cruz), anti Cx43 (Sigma) or antismooth muscle actin (Beckman Coulter, Immunotech) overnight at 4°C, followed by incubation with a fluorescent antibody.

Statistical Analysis
Values are given as mean±SEM. Differences between groups were analyzed by ANOVA and Bonferoni’s adjustment for multiple comparisons. In the isolated perfused kidney experiments, the three values obtained within an experimental period were averaged and used for statistical analysis. Student’s paired t-test was used to calculate levels of significance within individual kidneys.

Probability values less than 0.05 were considered statistically significant


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Homozygous (Cx40+/–) but not heterozygous Cx40 (Cx40+/–) deficient mice are hypertensive (Figure 1, upper panel). Surprisingly, the steady state levels of plasma renin concentration and of renal renin mRNA expression were 5.1-fold and 2.4-fold increased in Cx40–/– mice relative to their WT controls (Cx40+/+) (Figure 1, middle and lower panel), whereas Cx40+/– mice behaved like WT controls. Renal renin content as a measure for stored renin was increased by 80% in Cx40–/– and was normal relative to wild-types in Cx40+/– mice (Table 1). Because hypertension in association with a high renin status indicates a renin-dependent hypertension, the effect of Ang I-converting-enzyme (ACE) inhibition was examined. ACE inhibition using enalapril (10 mg/kg per day) applied for 5 days lowered systolic blood pressure on average by 14 mm Hg in Cx40+/+ and Cx40+/– and by 21 mm Hg in Cx40–/– mice but did not correct blood pressure of Cx40–/– mice to the values observed in WT mice (Figure 1, upper panel), suggesting the existence of renin/Ang II-dependent and -independent components of hypertension in Cx40-deficient animals.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Figure 1. Effects of ACE-inhibition using enalapril on systolic blood pressure (upper panel), plasma renin concentration (middle panel), and renin mRNA expression (lower panel) in connexin40 knockout mice (Cx40–/–) in heterozygous Cx40 deficient mice (Cx40+/–) and their WT controls (Cx40+/+). * P<0.05 vs vehicle.


View this table:
[in this window]
[in a new window]

  		Table 1. Renal Renin Content (ng Ang I/mgxh)

In view of this, we hypothesized that the physiologic negative feedback control of renin secretion and renin gene expression exerted by intrarenal pressure, Ang II or sodium load might be defective in Cx40-deficient mice.

As shown in Figure 1 ACE-inhibition increased plasma renin concentration (PRC) {approx}7-fold and renal renin mRNA levels {approx}5-fold in Cx40+/+ and Cx40+/– mice, while renin mRNA increased only 2-fold and PRC did not change significantly in Cx40–/– mice. This difference was further corroborated by experiments in isolated perfused kidneys obtained from WT and Cx40–/– mice (Figure 2). The inhibitory action of Ang II (10 pmol/L – 1 nmol/L) on renin secretion was clearly impaired in Cx40–/– kidneys, because the concentration response curve was significantly shifted to higher concentrations in Cx40-deficient kidneys (IC50 Cx40–/–: 240nmol/L; Cx40+/+: 35nmol/L; Figure 2, upper panel). This was not because of an altered vasoconstrictor effect of Ang II because the decrease of perfusate flow at constant perfusion pressure was rather similar in both genotypes (Figure 2, lower panel).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Figure 2. Effects of Ang II on renin secretion rates (upper panel) and perfusate flow (lower panel) of isolated perfused kidneys of Cx40 knockout mice (Cx40–/–, white circles) and their WT controls (Cx40+/+, black circles). *P<0.05 vs Cx40+/+; n=5 each genotype.

We next studied the negative feedback exerted by intrarenal pressure on renin secretion and synthesis. The classic maneuver to examine the pressure control of the renin system in vivo is experimental unilateral renal stenosis, which stimulates renin secretion in the hypoperfused kidney, leads to renin dependent hypertension and thus suppresses renin secretion in the contralateral untouched kidney. We therefore clipped the left renal arteries of WT and Cx40-deficient mice with a 0.11 mm (inner diameter) clip and analyzed the animals seven days after placing the clips. At that time point a clear weight difference between clipped and nonclipped kidneys had developed (right/left ratio in wild type: 1.02±0.1 for sham, 1.52±0.2 for clipped; ratio in Cx40-deficient mice: 1.05±0.1 for sham, 1.47±0.25 for clipped), indicating the efficacy of the clipping maneuver in both genotypes. In WT mice renal artery clipping led to a 3.4-fold increase of plasma renin concentration, to a 8.2-fold side difference of renin mRNA between left and right kidneys and to a 32 mm Hg increase of systolic blood pressure (Figure 3). In Cx40 deficient mice, left renal artery clipping did not change plasma renin concentration or blood pressure and there was only a minor 2.2-fold side difference in renin mRNA levels (Figure 3), suggesting that the stimulation of the renin system by a decrease of intrarenal blood pressure was widely abrogated in Cx40-deficient mice. In kidneys isolated from WT mice renin secretion was clearly pressure dependent because decreases of perfusion pressure stimulated and increases of perfusion pressure inhibited renin secretion (Figure 4), thus establishing the well known inverse relationship between renal perfusion pressure and renin secretion. This classic inverse relationship between perfusion pressure and renin secretion was most strikingly changed in Cx40 deficient kidneys (Figure 4, upper panel) or in WT kidneys treated with the gap junction inhibitor 18{alpha}-glycyrrhetinic acid 10 µmol/L (18{alpha}-GA, Figure 4, lower panel), because renin secretion increased in parallel with perfusion pressure. Such a positive link between perfusion pressure and renin secretion can also be established in normal, WT kidneys by lowering the extracellular calcium concentration toward the micromolar range (Figure 4, lower panel). Notably, glycyrrhizic acid (GZA) an inactive analogue of 18{alpha}-glycyrrhetinic acid did not significantly alter renin release rates (Figure 4, lower panel).


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Figure 3. Effects of clipping the left renal artery on systolic blood pressure (upper panel), plasma renin concentration (middle panel), and renin mRNA expression (lower panel) in Cx40 knockout mice (Cx40–/–) and their WT controls (Cx40+/+). *P<0.05 vs sham; #P<0.01 vs sham.


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Figure 4. Upper panel: Perfusion pressure dependent regulation of renin secretion rates from kidneys of Cx40 knockout mice (Cx40–/–) and their WT (Cx40+/+). Lower panel, Effects of the gap junction inhibitor 18{alpha}-glycyrrhetinic acid (18{alpha}GA) 10µmolL, its inactive analogue glycyrrhizic acid (GZA) 10µmol/L or a reduction of a reduction of the extracellular calcium concentration by adding EGTA 3.1 mmol/L to the perfusate (EGTA) on the pressure dependent regulation of renin secretion from kidneys of WT mice. For comparison the data of Cx40 knockout mice without any treatment shown in the upper panel are depicted (control). *P<0.05 vs 90 mmHg; n=5 each genotype/treatment.

In line, addition of the calcium chelator EGTA (3.1 mmol/L) to the perfusate caused a 6-fold increase of renin secretion from WT kidneys, whereas this increase was completely abolished in Cx40 deficient kidneys (Figure 5, upper panel).


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5. Upper panel, Renin secretion rates from isolated perfused kidneys of Cx40 knockout mice (Cx40–/–) and their WT (Cx40+/+) under baseline conditions (control) as well as after stimulation with isoproterenol 10nmol/L or after lowering the extracellular calcium concentration in the submicromolar range (EGTA). *P<0.05 vs control, n=5 each genotype and treatment. Lower panel, Renin release from isolated juxtaglomerular cells of primary cultures of Cx40 knockout mice (Cx40–/–) and their WT (Cx40+/+). Cells were treated with forskolin 5 µmol/L alone (forskolin) or with a combination of forskolin with either Ang II 1µmol/L or thapsigargin 1 µmol/L. #P<0.001 vs control, §P<0.05 vs forskolin alone; n=4 each genotype.

At the same time, ß-adrenergic stimulation (isoproterenol, 10 nmol/L) strongly enhanced renin secretion in Cx40-deficient kidneys and this stimulus was equally efficient in both genotypes (Figure 5, upper panel). In addition, the direct stimulation of renin gene expression initiated by ß-adrenoreceptor activation via isoproterenol was not altered in Cx40 deficient animals in vivo (Cx40+/+ from 1.1+0.3 to 5.3+0.3 renin mRNA/actin mRNA, P<0.05; Cx40–/– from 4.3+0.4 to 13.4+3.4 renin mRNA/actin mRNA, P<0.05; data not shown).

In short term primary cultures of juxtaglomerular cells isolated from WT and from Cx40-deficient kidneys we found no difference in the secretory behavior of the 2 genotypes with regard to cAMP stimulation by forskolin nor with regard to the calcium dependent inhibition induced by Ang II or the SERCA-inhibitor thapsigargin. (Figure 5, lower panel).

We further examined the regulation of the renin system by salt intake in WT and Cx40-deficient mice because salt load controls the renin system by a supposedly different feedback mechanism. In both genotypes plasma renin concentration and renal renin mRNA levels were inversely related to the amount of salt intake and blood pressure remained unchanged in both groups of mice (Figure 6).


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		Figure 6. Effects of a low or a high salt intake on systolic blood pressure (upper panel), plasma renin concentration (middle panel), and renin mRNA expression (lower panel) in Cx40 knockout mice (Cx40–/–) and their WT controls (Cx40+/+). n=4 each genotype. *P<0.05 vs low salt

Finally, we considered the possibility that the different behavior of renin secretion in Cx40-deficient kidneys was because of the compensatory upregulation of other connexins rather than to the lack of Cx40. Coimmunolabeling for Cx40 and renin showed a good overlap of renin and Cx40 signals in the juxtaglomerular region in WT mice (Figure 7, A and B). Coimmunolabeling for Cx43 and renin showed no overlap of renin and Cx43 in the juxtaglomerular region neither of WT nor of Cx40 deficient mice. (Figure 7, C and D). We found Cx43 immunoreactivity in larger kidney vessels (Figure 7, E) and predominantly in the heart (Figure 7, F), indicating that the immunostaining procedure worked in general. The lack of upregulation of Cx43 in the juxtaglomerular region of Cx40-deficient kidneys was also confirmed by mRNA analysis and by western blotting using extracts from kidney cortex (please see the online data supplement available at http://circres.ahajournals.org).


Figure 7
View larger version (66K):
[in this window]
[in a new window]

  		Figure 7. Localization of renin (green), Cx40 and Cx43 (red) immunoreactivity in the kidney sections of Cx40–/– mice and their WT controls. Double staining for renin and Cx40 protein indicates the co expression of renin and Cx40 in juxtaglomerular cells of WT mice (A). No Cx40 staining was observed in Cx40–/– mice (B). Double staining for renin and Cx43 showed no overlap for both proteins in juxtaglomerular cells neither of WT (C) nor of Cx40 knockout mice (D). Arrows indicate renin immunoreactive areas (green). Cx43 (red) immunostaining were detected in the endothelium of large renal arterial vessels, labeled by {alpha}-smooth muscle actin staining (green) (E, arrows) and in the myocardium of the heart (F). Bar indicates 50 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Our findings indicate that the classic negative feedback loops which involve Ang II and the intrarenal blood pressure and which control the activity of the renin system, are disturbed in the absence of Cx40.

Although there is no doubt that Ang II can inhibit renin secretion at the level of the juxtaglomerular cells,1 as also confirmed in this study, the physiological relevance of such a "short-loop" feedback is meanwhile questioned. Studies with genetically engineered mice provided evidence that the normal feed-back control of renin secretion by Ang II in vivo is mainly mediated indirectly.23,24

The results of our study would fit with such an assumption of at least 2 inhibitory mechanisms mediating the action of Ang II. Firstly, a direct one at the level of juxtaglomerular cells, that occurs at higher concentrations of Ang II and that does not require Cx40 connexins. Secondly, an indirect one that occurs already at lower concentrations of Ang II and that requires either Cx40 hemi channels or cell to cell communication through Cx40 gap junctions. It has been postulated that changes of blood pressure may account for the indirect component in the feedback regulation of renin secretion by Ang II. Again this hypothesis would be fully supported by our data, as they show that pressure regulation of renin synthesis and renin secretion are abolished in the absence of Cx40 connexins. Because the pressure dependency of renin secretion was also abolished in the presence of a pharmacological gap junction blocker our findings strongly suggest that cell to cell communication via Cx40 gap junctions are essentially involved in the renal "barosensor" mechanism controlling renin synthesis and secretion

Based in the immunohistological data available it is conceivable that within the afferent arterioles of the kidney gap junctions formed by Cx40 establish a functional syncytium and connect the endothelial cells to the renin secreting cells and these possibly to extra- and intraglomerular mesangial cells.9–12 Therefore, apart from renin secreting cells themselves also mesangial and endothelial cells could act as barosensors in principle. The feasibility of endothelial cells to act as mechanosensors affecting the function of the underlying smooth muscle cell layer is well established.25,26 For example, the electrotonic spread of an endothelial hyperpolarization to the adjacent smooth muscle is dependent on myoendothelial gap junctions.27,28 Such a mechanism may also be active to attenuate renin secretion in the juxtaglomerular cells. Interestingly, it has been shown recently that the release of NO and the cytosolic calcium concentration in endothelial cells of afferent arterioles are dependent on intravasal pressure,29 supporting the idea that endothelial cells can function as primary mechanosensors. Moreover, a close spatial contact of renin secreting cells with endothelial cells in fact modulates renin secretion.30

A common characteristic of the inhibitory effects of Ang II and of perfusion pressure on renin secretion is their dependency on extracellular calcium, suggesting that both parameters involve a calcium dependent step in their signaling pathways.3,4 In fact, it is commonly assumed that a rise of the cytosolic calcium concentration inhibits renin secretion and also renin gene expression at the level of juxtaglomerular cells,4,31 a phenomenon that is termed "calcium paradox" of renin.

Notably, in the absence of Cx40 renin secretion behaved as if the extracellular calcium concentration was low, as supported by 3 lines of evidence. Firstly, lowering of the calcium concentration in the perfusate did not stimulate renin secretion in Cx40-deficient kidneys as usually expected (Figure 5). Secondly, pressure dependent renin secretion in Cx40 deficient kidneys was similar to that of Cx40 expressing kidneys at low extracellular calcium concentration (Figure 3). Finally, the concentration-dependent inhibition of Ang II on renin secretion was shifted to the right, which can also be achieved by lowering the extracellular calcium concentration.4

Because the conductance of gap junctions is inversely related to the cytosolic calcium concentration ie, conductivity is enhanced by low calcium32 it is unlikely, that the effect of low extracellular calcium was because of a decoupling of gap junctions thus mimicking Cx40 deficiency.

In contrast, it is tempting to speculate from our findings that Cx40 is important for the induction and/or propagation of cytosolic calcium increases in renin secreting cells. As already mentioned previously there is good evidence that gap junctions are supporting the spreading of calcium waves from cell to cell, thus synchronizing the activity of tissues such as vascular smooth muscle cells or endocrine cells.33–37

It has been reported recently that the substitution of Cx43, which is not expressed in renin producing cells,10–12,14 by Cx32 is associated with diminished renin synthesis and renin secretion under stimulatory conditions15 and the authors proposed that Cx 43 is required for a normal responsiveness of the renin system on pressure decreases.15 Because Cx43 expression in the aorta is compensatorily increased in Cx40 deficient mice,38 it was to rule out that a compensatory increase of Cx43 in renin producing cells of Cx40 deficient mice might account for the dysregulation of the renin system as observed in this study. In accordance with the results of others10–12,14,39 we found Cx43 immunoreactivity in larger kidney vessels and in the heart but not in renin producing cells of WT or Cx40-deficient mice. Moreover, neither Western blot analysis nor mRNA analysis gave any evidence for an upregulation of Cx43 expression in Cx40 deficient mice. Therefore, we come to the conclusion that a possible upregulation of Cx43 in renin producing cells does not contribute to the dysregulation of the renin system in mice lacking Cx40.

In summary, our data provide first evidence that Cx40 connexins are required to exert an inhibition of renin secretion and synthesis in the kidney, because Ang II and intrarenal pressure which both act as physiologic negative feedback regulators loose their capability to attenuate renin secretion in the absence of Cx40. It remains to be elucidated if such a pathomechanism accounts for a small fraction of essential hypertension in humans. Interestingly, a polymorphism within the Cx40 promotor has been demonstrated to be associated with an increased risk of hypertension in men.40

Our findings could provide a plausible explanation for the combination of a high renin status with high blood pressure in Cx40 deficient mice, without renin being the only cause for hypertension. As already proposed previously an altered vasomotion of arterioles may contribute to the hypertension observed in Cx40-deficient mice.13,17,41


*    Acknowledgments
 
Breeder pairs of Cx40 knockout mice were kindly provided by Professor K. Willecke, Institut für Genetik, University of Bonn, Germany.

Sources of Funding

The study was financially supported by the Deutsche Forschungsgemeinschaft (DFG) (Sonderforschungsbereich 699 to C.W., A.K., and F.S.; DFG Wi 2071/1–1 to C.d.W.).

Disclosures

None.


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received May 29, 2006; resubmission received July 25, 2006; revised resubmission received December 20, 2006; accepted January 17, 2007.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Wagner C, Kurtz A. Regulation of renal renin release. Curr Opin Nephrol Hypertens. 1998; 7: 437–441.[Medline] [Order article via Infotrieve]
  2. Hackenthal E, Paul M, Ganten D, Taugner R. Morphology, physiology, and molecular biology of renin secretion. Physiol Rev. 1990; 70: 1067–1116.[Free Full Text]
  3. Scholz H, Hamann M, Gotz KH, Kurtz A. Role of calcium ions in the pressure control of renin secretion from the kidneys. Pflugers Arch. 1994; 428: 173–178.[CrossRef][Medline] [Order article via Infotrieve]
  4. Schweda F, Kurtz A. Cellular mechanism of renin release. Acta Physiol Scand. 2004; 181: 383–390.[CrossRef][Medline] [Order article via Infotrieve]
  5. Taugner R, Kirchheim H, Forssmann WG. Myoendothelial contacts in glomerular arterioles and in renal interlobular arteries of rat, mouse and Tupaia belangeri. Cell Tissue Res. 1984; 235: 319–325.[Medline] [Order article via Infotrieve]
  6. Taugner R, Buhrle CP, Nobiling R. Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice. Cell Tissue Res. 1984; 237: 459–472.[Medline] [Order article via Infotrieve]
  7. Taugner R, Schiller A, Kaissling B, Kriz W. Gap junctional coupling between the JGA and the glomerular tuft. Cell Tissue Res. 1978; 186: 279–285.[Medline] [Order article via Infotrieve]
  8. Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res. 2004; 62: 228–232.[CrossRef][Medline] [Order article via Infotrieve]
  9. Hwan Seul K, Beyer EC. Heterogeneous localization of connexin40 in the renal vasculature. Microvasc Res. 2000; 59: 140–148.[CrossRef][Medline] [Order article via Infotrieve]
  10. Haefliger JA, Demotz S, Braissant O, Suter E, Waeber B, Nicod P, Meda P. Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension. Kidney Int. 2001; 60: 190–201.[CrossRef][Medline] [Order article via Infotrieve]
  11. Arensbak B, Mikkelsen HB, Gustafsson F, Christensen T, Holstein-Rathlou NH. Expression of connexin37, 40, and 43 mRNA and protein in renal preglomerular arterioles. Histochem Cell Biol. 2001; 115: 479–487.[Medline] [Order article via Infotrieve]
  12. Zhang J, Hill CE. Differential connexin expression in preglomerular and postglomerular vasculature: accentuation during diabetes. Kidney Int. 2005; 68: 1171–1185.[CrossRef][Medline] [Order article via Infotrieve]
  13. de Wit C, Roos F, Bolz SS, Pohl U. Lack of vascular connexin40 is associated with hypertension and irregular arteriolar vasomotion. Physiol Genomics. 2003; 13: 169–177.[Abstract/Free Full Text]
  14. Barajas L, Liu L, Tucker M. Localization of connexin43 in rat kidney. Kidney Int. 1994; 46: 621–626.[Medline] [Order article via Infotrieve]
  15. Haefliger JA, Krattinger N, Martin D, Pedrazzini T, Capponi A, Doring B, Plum A, Charollais A, Willecke K, Meda P. Connexin43-dependent mechanism modulates renin secretion and hypertension. J Clin Invest. 2006; 116: 405–413.[CrossRef][Medline] [Order article via Infotrieve]
  16. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr Biol. 1998; 8: 299–302.[CrossRef][Medline] [Order article via Infotrieve]
  17. de Wit C, Roos F, Bolz SS, Kirchhoff S, Kruger O, Willecke K, Pohl U. Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. Circ Res. 2000; 86: 649–655.[Abstract/Free Full Text]
  18. Morello F, de Boer RA, Steffensen KR, Gnecchi M, Chisholm JW, Boomsma F, Anderson LM, Lawn RM, Gustafsson JK, Lopez-Ilasaca M, Pratt RE, Dzau VJ. Liver X receptors alpha and beta regulate renin expression in vivo. J Clin Invest. 2005; 115: 1913–1922.[CrossRef][Medline] [Order article via Infotrieve]
  19. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162: 156–159.[Medline] [Order article via Infotrieve]
  20. Schweda F, Wagner C, Kramer BK, Schnermann J, Kurtz A. Preserved macula densa-dependent renin secretion in A1 adenosine receptor knockout mice. Am J Physiol Renal Physiol. 2003; 284: F770–777.[Abstract/Free Full Text]
  21. Della Bruna R, F.Pinet, P.Corvol, and AKurtz. Regulation of renin secretion, and renin synthesis by second messenger in isolated mouse juxtaglomerular cells. Cell Physiol Biochem. 1991; 1: 98–110.
  22. Mann B, Hartner A, Jensen BL, Hilgers KF, Hocherl K, Kramer BK, Kurtz A. Acute upregulation of COX-2 by renal artery stenosis. Am J Physiol Renal Physiol. 2001; 280: F119–F125.[Abstract/Free Full Text]
  23. Matsusaka T, Nishimura H, Utsunomiya H, Kakuchi J, Niimura F, Inagami T, Fogo A, Ichikawa I. Chimeric mice carrying ‘regional’ targeted deletion of the angiotensin type 1A receptor gene. Evidence against the role for local angiotensin in the in vivo feedback regulation of renin synthesis in juxtaglomerular cells. J Clin Invest. 1996; 98: 1867–1877.[Medline] [Order article via Infotrieve]
  24. Crowley SD, Gurley SB, Oliverio MI, Pazmino AK, Griffiths R, Flannery PJ, Spurney RF, Kim HS, Smithies O, Le TH, Coffman TM. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. J Clin Invest. 2005; 115: 1092–1099.[CrossRef][Medline] [Order article via Infotrieve]
  25. Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature. 2005; 437: 426–431.[CrossRef][Medline] [Order article via Infotrieve]
  26. Lehoux S, Castier Y, Tedgui A. Molecular mechanisms of the vascular responses to haemodynamic forces. J Intern Med. 2006; 259: 381–392.[CrossRef][Medline] [Order article via Infotrieve]
  27. Sandow SL, Hill CE. Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses. Circ Res. 2000; 86: 341–346.[Abstract/Free Full Text]
  28. Griffith TM. Endothelium-dependent smooth muscle hyperpolarization: do gap junctions provide a unifying hypothesis? Br J Pharmacol. 2004; 141: 881–903.[CrossRef][Medline] [Order article via Infotrieve]
  29. Pittner J, Wolgast M, Casellas D, Persson AE. Increased shear stress-released NO and decreased endothelial calcium in rat isolated perfused juxtamedullary nephrons. Kidney Int. 2005; 67: 227–236.[CrossRef][Medline] [Order article via Infotrieve]
  30. Kurtz A, Kaissling B, Busse R, Baier W. Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells. J Clin Invest. 1991; 88: 1147–1154.[Medline] [Order article via Infotrieve]
  31. Klar J, Sigl M, Obermayer B, Schweda F, Kramer BK, Kurtz A. Calcium inhibits renin gene expression by transcriptional and posttranscriptional mechanisms. Hypertension. 2005; 46: 1340–1346.[Abstract/Free Full Text]
  32. Peracchia C. Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim Biophys Acta. 2004; 1662: 61–80.[Medline] [Order article via Infotrieve]
  33. Haddock RE, Hill CE. Rhythmicity in arterial smooth muscle. J Physiol. 2005; 566: 645–656.[Abstract/Free Full Text]
  34. Gaspers LD, Thomas AP. Calcium signaling in liver. Cell Calcium. 2005; 38: 329–342.[CrossRef][Medline] [Order article via Infotrieve]
  35. Michon L, Nlend Nlend R, Bavamian S, Bischoff L, Boucard N, Caille D, Cancela J, Charollais A, Charpantier E, Klee P, Peyrou M, Populaire C, Zulianello L, Meda P. Involvement of gap junctional communication in secretion. Biochim Biophys Acta. 2005; 1719: 82–101.[Medline] [Order article via Infotrieve]
  36. Serre-Beinier V, Mas C, Calabrese A, Caton D, Bauquis J, Caille D, Charollais A, Cirulli V, Meda P. Connexins and secretion. Biol Cell. 2002; 94: 477–492.[CrossRef][Medline] [Order article via Infotrieve]
  37. Davies PF, Zilberberg J, Helmke BP. Spatial microstimuli in endothelial mechanosignaling. Circ Res. 2003; 92: 359–370.[Abstract/Free Full Text]
  38. Kruger O, Beny JL, Chabaud F, Traub O, Theis M, Brix K, Kirchhoff S, Willecke K. Altered dye diffusion and upregulation of connexin37 in mouse aortic endothelium deficient in connexin40. J Vasc Res. 2002; 39: 160–172.[CrossRef][Medline] [Order article via Infotrieve]
  39. Hill CE, Phillips JK, Sandow SL. Heterogeneous control of blood flow amongst different vascular beds. Med Res Rev. 2001; 21: 1–60.[CrossRef][Medline] [Order article via Infotrieve]
  40. Firouzi M, Kok B, Spiering W, Busjahn A, Bezzina CR, Ruijter JM, Koeleman BP, Schipper M, Groenewegen WA, Jongsma HJ, de Leeuw PW. Polymorphisms in human connexin40 gene promoter are associated with increased risk of hypertension in men. J Hypertens. 2006; 24: 325–330.[Medline] [Order article via Infotrieve]
  41. Rummery NM, Hill CE. Vascular gap junctions and implications for hypertension. Clin Exp Pharmacol Physiol. 2004; 31: 659–667.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
F. Hanner, J. von Maltzahn, S. Maxeiner, I. Toma, A. Sipos, O. Kruger, K. Willecke, and J. Peti-Peterdi
Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R371 - R380.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
I. Toma, E. Bansal, E. J. Meer, J. J. Kang, S. L. Vargas, and J. Peti-Peterdi
Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2008; 294(6): R1769 - R1776.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
T. Takenaka, T. Inoue, Y. Kanno, H. Okada, C. E. Hill, and H. Suzuki
Connexins 37 and 40 transduce purinergic signals mediating renal autoregulation
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R1 - R11.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. E. Wolfle, V. J. Schmidt, B. Hoepfl, A. Gebert, S. Alcolea, D. Gros, and C. de Wit
Connexin45 Cannot Replace the Function of Connexin40 in Conducting Endothelium-Dependent Dilations Along Arterioles
Circ. Res., December 7, 2007; 101(12): 1292 - 1299.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. Wagner, C. de Wit, M. Gerl, A. Kurtz, and K. Hocherl
Increased expression of cyclooxygenase 2 contributes to aberrant renin production in connexin 40-deficient kidneys
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293(5): R1781 - R1786.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
F. Schweda, U. Friis, C. Wagner, O. Skott, and A. Kurtz
Renin Release
Physiology, October 1, 2007; 22(5): 310 - 319.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
F. Lang, V. Vallon, M. Knipper, and P. Wangemann
Functional significance of channels and transporters expressed in the inner ear and kidney
Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1187 - C1208.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
100/4/556    most recent
01.RES.0000258856.19922.45v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wagner, C.
Right arrow Articles by Schweda, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation