Reciprocal expression of connexin 40 and 45 during phenotypical changes in renin-secreting cells

Abstract

Gap junctional coupling of renin-producing cells is of major functional importance for the control of renin synthesis and release. This study was designed to determine the relevance of the vascular gap junction protein connexin 45 (Cx45) for the control of renin expression and secretion. By crossbreeding mice which drive Cre recombinase under the control of the endogenous renin promoter with mice harboring floxed Cx45 gene alleles, we generated viable mice with a deletion of Cx45 in the renin cell lineage. These mice were normotensive, and renin cells in their kidneys were normal with regard to localization and number. Sodium deficiency induced typical recruitment of renin-producing cells along afferent arterioles, whereas sodium overload resulted in a decrease in the number of cells expressing renin. Regulation of renin secretion by perfusion pressure, catecholamines, and angiotensin II from isolated kidneys of mice with renin cell-specific deletion of Cx45 was normal. Analyzing Cx45 promoter activity in cells of the preglomerular arteriolar tree by using mice driving the reporter gene LacZ under the control of the Cx45 promoter revealed strong staining in smooth muscle cells of the media, whereas renin-expressing cells were almost devoid of LacZ staining. Conversely, renin-producing cells, but not vascular smooth muscle cells expressed the gap junction protein Cx40. These findings suggest that Cx45 plays no major functional role in renin-producing cells, probably because the expression of Cx45 is downregulated in these cells. Since renin-producing cells in the adult kidney can reversibly transform into vascular smooth muscle cells and vice versa, our findings on connexin expression indicate that these phenotype switches are paralleled by characteristic reciprocal changes in the transcriptional activity of Cx40 and Cx45 genes.

gap junctional coupling of renin-secreting cells in the kidney is important for the correct position of renin-secreting cells and for the control of renin secretion. So far, evidence has been provided that the gap junction protein connexin 40 (Cx40) but not Cx37 is highly relevant in this respect (8, 21, 22, 24). In addition, a functional role of Cx45, which is typical for preglomerular smooth muscle cells in the kidney, has been suggested from experiments with mice bearing the Cx45 gene deletion in the nestin-expressing cell lineage (7). In the adult kidney, preglomerular vascular smooth muscle cells can, in fact, reversibly transform into renin-expressing cells under situations in which the sodium homeostasis of the adult organism is threatened (2, 6, 18). Since afferent arteriolar smooth muscle cells and renin-expressing cells communicate with each other via gap junctions (1, 5, 15, 19), a role of Cx45 in this communication is therefore conceivable. Immunohistochemical investigations have so far failed to localize Cx45 in the plasma membrane of typical juxtaglomerular renin-producing cells (10). This failure might be explained by a low density of Cx45 gap junctions in the plasma membrane of renin-producing cells. Conversely, the alterations of renin secretion in mice lacking Cx45 in the nestin-expressing cell lineage (7) could have resulted from indirect effects on renin-producing cells. We wished to define the functional relevance of Cx45 in renin expression and distribution under basal conditions and under conditions in which preglomerular smooth muscle cells transform into renin-expressing cells. Because mice with global deletion of Cx45 are not viable (9), we used a Cre-lox approach to disrupt the Cx45 gene in the renin cell lineage and simultaneously we examined the activity of the Cx45 promoter in renin-producing cells in situ.

MATERIALS AND METHODS

Animals.

All animal experiments were conducted according to the National Institutes of Health (NIH) guidelines for the care and use of animals in research. The experiments were approved by a local ethics committee. The renin-Cre:Cx45^fl/fl mice were developed from two mouse strains: mice in which coding exon three of the Cx45 gene was flanked by loxP sites (Cx45^fl/fl) (13) and mice with targeted insertion of Cre recombinase into the Ren1d locus (Ren1d^+/Cre, renin-Cre) (17). After Cre recombinase activity, an enhanced green fluorescent protein (eGFP) reporter gene is expressed under the control of the endogenous Cx45 promoter.

Animal treatment protocol.

Male renin-Cre:Cx45^fl/fl and their controls (Cx45^fl/fl) were distributed to three experimental groups in each case. The groups were treated as follows.

Group I: mice were fed a normal-salt diet (NaCl 0,6%) over a period of 21 days.

Group II: mice were fed a high-salt diet (NaCl 4%) over a period of 21 days.

Group III: mice were fed a low-salt diet (NaCl 0,02%) over a period of 21 days and also received the angiotensin-converting enzyme (ACE) inhibitor enalapril (on average 10 mg·kg⁻¹·day⁻¹) via their drinking water for the last 15 days of the experiment.

After treatment, blood samples (50 μl) for the determination of plasma renin concentration were taken from the tail vein of the renin-Cre:Cx45^fl/fl mice and their controls. After the experimental periods, the animals were deeply anesthetized with sevoflurane and euthanized using cervical dislocation. Kidneys were removed rapidly and either frozen and stored in liquid nitrogen for further determination of the mRNA concentration or cryoconserved for the preparation of cryosections for Cx40- and Cx45-renin costaining. For the preparation of paraffin sections and LacZ staining sections, the kidneys were perfusion-fixed with 4% paraformaldeyhde.

Blood pressure measurements.

Systolic blood pressure was measured noninvasively by the tail-cuff method. Mice were put in a steel cover on a 30°C prewarmed platform and habituated to the experimental procedure for 5 subsequent days. Blood pressure was determined on 10 subsequent days.

Isolated, perfused mouse kidney model.

Kidneys of Cx45^fl/fl and renin-Cre:Cx45^fl/fl mice were perfused as described in Schweda et al. (16). Briefly, the animals were anesthetized with an intraperitoneal injection of 12 mg/kg xylazine and 80 mg/kg ketamine-HCl, the abdominal aorta was cannulated, and the right kidney was excised, placed in a thermostated moistening chamber, and perfused at constant pressure (90 mmHg). Using electronic feedback control, perfusion pressure could be changed and held constant in a pressure range between 40 and 140 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 bovine serum albumin and with freshly washed human red blood cells (a 10% hematocrit).

Stock solutions of angiotensin II or isoproterenol were dissolved in freshly prepared perfusate and infused into the arterial limb of the perfusion circuit. For lowering the extracellular calcium concentration into the submicromolar range, the calcium chelator EGTA (3.12 mM) was added to the perfusate.

For the determination of renin secretion rates, three samples of the venous effluent were taken in intervals of 2 min during each experimental period. Renin activity in the venous effluent was determined by radioimmunoassay (Byk & DiaSorin Diagnostics, Dietzenbach, Germany) as described previously (16). Renin secretion rates were calculated as the product of the arteriovenous differences of renin activity and the perfusate flow rate (ml·min⁻¹·g kidney wt⁻¹).

Microdissection of glomeruli.

Glomeruli with attached afferent arterioles for the determination of the efficacy of the renin-Cre Cx45-flox recombination by eGFP-fluorescence were obtained by collagenase digestion protocol as described previously (20).

Determination of mRNA expression levels by real-time PCR.

Total RNA was isolated from kidneys as described by Chomczynski and Sacchi (4) and quantified by a photometer. The cDNA was synthesized by Moloney murine leukemia virus RT (Superscript, Invitrogen) and amplified with primers as described before (10, 12). For quantification of mRNA expression, real-time PCR was performed using a Light Cycler Instrument and the LightCycler 480 SYBR Green I Master kit (Roche Diagnostics) and GAPDH as a control.

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 h at 37°C, with plasma from bilaterally nephrectomized male rats as the renin substrate (22). The generated angiotensin I (ng·ml⁻¹·h⁻¹) was determined using radioimmunoassay (Byk & DiaSorin Diagnostics).

Antibodies.

Primary antibodies used for immunohistochemistry were anti-Cx40 (Biotrend), anti-renin (Davids Biotechnologie), anti-α-smooth muscle actin (anti-α-SMA; Beckman Coulter Immunotech), and anti-SM22 (Abcam). Cy2-, tetramethyl-rhodamine isothiocyanate (TRITC)-, and Cy5-conjugated secondary antibodies were purchased from Dianova.

Cx45-LacZ and renin costaining.

Kidneys from Cx45^+/LacZ mice were perfusion-fixed with 4% paraformaldeyhde, embedded in Tissue-Tec, frozen on dry ice, sectioned (5 μm) on a cryostat, and transferred onto Superfrost plus slides. Sections were rinsed three times in LacZ washing buffer (0.1 M phosphate buffer pH 7.4, 1.25 mM MgCl₂, 5 mM EGTA, 0.2% Nonidet P-40, and 0.01% sodium deoxycholate), and stained in LacZ substrate buffer (LacZ washing buffer supplemented with 0.5 mg/ml X-Gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide) overnight at 37°C. Sections were then washed three times in PBS and blocked in a buffer containing PBS, 1% BSA, and 10% horse serum for 30 min. The anti-renin antibody (1:200) was diluted in the same blocking solution, and sections were then incubated at 4°C overnight. The next day, sections were washed three times in PBS containing 1% BSA and incubated with a Cy2-conjugated secondary antibody (1:400) for 90 min at room temperature. After washing in PBS, sections were mounted with DakoCytomation Glycergel mounting medium and viewed with an Axiovert Microscope (Zeiss, Jena, Germany).

Cx40, renin, and SM22 costaining.

Kidneys were frozen unfixed in Tissue-Tek and sectioned at 5 μm with a cryostat. Without further storing, sections were fixed in 1% paraformaldehyde at room temperature for 1 min, washed three times in Tris-buffered saline (TBS), and then blocked in TBS supplemented with 5% horse serum and 0.3% Triton X-100 (TTBS) for 2 h. Primary antibodies were diluted in TTBS using anti-Cx40 (1:200), anti-renin (1:200), and anti-SM22 (1:200), incubating sections at 4°C overnight. On the next day, sections were washed three times in TBS before incubation with Cy2-, TRITC-, and Cy5-conjugated secondary antibodies in TTBS for 90 min. After three final washes in TBS, slices were mounted with DakoCytomation Glycergel mounting medium and viewed with an Axiovert Microscope.

Renin and α-SMA costaining.

Kidneys were perfusion-fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Immunolabeling was performed on 5-μm paraffin sections. After blocking with 10% horse serum and 1% BSA in PBS, sections were incubated with anti-renin and anti-α-SMA (1:10) antibodies overnight at 4°C, followed by incubation with Cy2- and TRITC-conjugated secondary antibodies. Slices were mounted with DakoCytomation Glycergel mounting medium and viewed with an Axiovert Microscope.

Statistical analysis.

All data are presented as means ± SE. Differences between groups were analyzed by analysis of variance and Bonferroni's adjustment. P values <0.05 were considered statistically significant.

RESULTS

Efficacy of renin-Cre Cx45-flox recombination.

The efficacy of the renin-Cre Cx45-flox recombination was assessed by the genotyping protocol, which produces an additional amplicon of the recombined gene (Fig. 1A) and by the appearance of eGFP-expressing cells in the preglomerular vasculature. Recombination will lead to a deletion of the Cx45 coding region and to the expression of an eGFP gene at the Cx45 gene locus, which was, in fact, observed (Fig. 1B). We therefore assumed that the Cx45 gene was substantially disrupted in the renin cell lineage.

Fig. 1.

Efficacy of the renin-Cre Cx45-flox recombination. A: the 620-bp PCR amplicon represents a Cx45 knockout allele and thus the efficacy of the renin-Cre Cx45-flox recombination in the kidney. This PCR product is seen only in Cx45^fl/fl mice harboring the renin-Cre allele. BI: microdissected glomerulum with an afferent arteriole. II: enhanced green fluorescent protein (eGFP) expression (green) in the afferent arteriole indicates Cx45 gene expression. Renin-Cre excised the floxed Cx45 coding region and placed the eGFP reporter under the control of the Cx45 promoter. Bar = 50 μm. Arrows indicate the afferent arteriole, and asterisks indicate the glomerulum.

Structural and functional consequences of Cx45 gene disruption in the renin cell lineage.

Renin-Cre:Cx45^fl/fl mice had normal blood pressure (systolic blood pressure of the genotypes: Cx45^fl/fl, 135.1 ± 5.7 mmHg; renin-Cre:Cx45^fl/fl, 132.4 ± 7.6 mmHg) and normal renin concentration in the plasma (Fig. 2A). Similarly, renin mRNA abundance was not different between control and renin-Cre:Cx45^fl/fl kidneys (Fig. 2B). Immunohistological analysis revealed that renin-expressing cells in renin-Cre:Cx45^fl/fl kidneys (Fig. 3C) were similar in number and localization to Cx45^fl/fl kidneys (Fig. 3A). Renin-expressing cells were located in the media layer of afferent arterioles at the entrance to the glomeruli.

Fig. 2.

Plasma renin concentration and renin mRNA abundance in Cx45^fl/fl and renin-Cre:Cx45^fl/fl mice under normal conditions or after treatment with a low-salt diet in combination with enalapril. A: plasma renin concentration in samples, taken from the tail vein. B: renin mRNA abundance is given relative to levels measured in Cx45^fl/fl mice on a normal-salt diet. Data are means ± SE of 5 kidneys of each genotype. *P < 0.05 vs. respective controls under normal conditions.

Fig. 3.

Localization of renin-expressing cells by immunoreactivity in kidney sections of Cx45^fl/fl and renin-Cre:Cx45^fl/fl mice during a normal-salt diet or after a low-salt diet combined with enalapril. Immunohistochemistry is shown of renin (green) and α-smooth muscle actin (SMA; red) in the control (a, normal-salt diet; b, low-salt diet/enalapril) and renin-Cre:Cx45^fl/fl mice (c, normal-salt diet; d, low-salt diet/enalapril). Bar = 50 μm. Asterisks indicate the glomeruli, and arrowheads indicate renin-producing cells. Five kidneys of each genotype have been analyzed. There is no difference in the number and the localization of the renin-expressing cells between the 2 genotypes.

The control of renin secretion from isolated kidneys by the β-receptor activator isoproterenol and by angiotensin II showed no difference between the two genotypes (Fig. 4A). The control of renin secretion by renal perfusion pressure was also normal in renin-Cre:Cx45^fl/fl kidneys (Fig. 4B).

Fig. 4.

A: effects of angiotensin II on renin secretion from isolated perfused kidneys of Cx45^fl/fl and renin-Cre:Cx45^fl/fl mice. Shown is angiotensin II-dependent regulation of renin secretion. Dose-response curves were performed at a pressure of 90 mmHg in the presence of isoproterenol (10 nM). B: pressure-dependent renin secretion in isolated perfused kidneys from renin-Cre:Cx45^fl/fl mice and their controls. Kidneys were perfused with a pressure from 40 to 140 mmHg in the presence of 10 nM isoproterenol. Data are means ± SE of 4 kidneys of each genotype. There was neither angiotensin II-dependent nor pressure-dependent difference in renin secretion rates between the 2 genotypes.

Challenge of renin expression induced by treatment of the animals with a combination of low salt and an ACE inhibitor increased plasma renin concentrations and renin gene expression in the two genotypes ∼20-fold (Cx45^fl/fl, 22.5-fold; renin-Cre:Cx45^fl/fl, 24-fold) (Fig. 2A) and 10-fold (Cx45^fl/fl, 10.5-fold; renin-Cre:Cx45^fl/fl, 9.9-fold) (Fig. 2B), respectively.

The increase in renin mRNA abundance went in parallel with a recruitment of renin cells along the preglomerular vessels, reflecting a transformation of vascular smooth muscle cells into renin-producing cells. This recruitment phenomenon was very similar in Cx45^fl/fl and in renin-Cre:Cx45^fl/fl mice (Fig. 3, B and D).

Cx45-LacZ and Cx40 expression in Cx45^+/LacZ mice.

Since the functional analysis as described above produced no evidence for a major direct function of Cx45 in renin-producing cells, we aimed to determine whether the promoter of the Cx45 gene is active at all in renin-producing cells. For this purpose, we analyzed Cx45^+/LacZ mice in which one copy of the Cx45 gene is replaced by the LacZ reporter gene, which is driven by the Cx45 promoter. Under basal conditions, the Cx45-LacZ was strongly expressed in the media layer of intrarenal arteries and arterioles and in disseminated intraglomerular cells. Renin-positive cells in the juxtaglomerular area displayed no LacZ staining (Fig. 5, A–C). Instead, the renin-producing cells expressed Cx40, while preglomerular vascular smooth muscle cells did not (Fig. 6, A and B).

Fig. 5.

Detection and localization of Cx45 gene promoter activity in kidney cortex of Cx45^+/LacZ mice by LacZ staining. Shown is LacZ (blue) and renin (green) staining in Cx45^+/LacZ mice on a normal-salt diet (b) or a low-salt diet in combination with enalapril (e). Also shown is a monochromatic view of Cx45-LacZ/renin staining in kidneys from Cx45^+/LacZ mice on a normal-salt diet (a and c) and on a low-salt diet/enalapril (d and f). To have better contrast, Cx45-LacZ staining is seen as dark. Bar = 50 μm. Arrowheads indicate cells that are negative for Cx45-LacZ staining, but positive for renin immunoreactivity, and asterisks indicate the glomeruli. Five kidneys of each genotype have been analyzed.

Fig. 6.

Cx40 immunostaining on kidney sections of Cx45^+/LacZ mice under normal conditions (a and b) and after treatment with a low-salt diet in combination with enalapril (c and d). Triple immunostaining is shown for Cx40 (red), renin (green), and SM22 (blue) in kidney of Cx45^+/LacZ mice on a normal-salt diet (a) and a low-salt diet in combination with enalapril (c). Also shown is a onochromatic view of Cx40 immunoreactivity in kidneys on a normal-salt (b) and on a low-salt diet in combination with enalapril (d). Bar = 50 μm. Arrows indicate Cx40 staining in endothelial cells, arrowheads indicate renin-producing cells, and asterisks mark the glomeruli. Five kidneys of each genotype have been analyzed. Cx40 is found in association with renin-expressing cells but not with smooth muscle cells of afferent arteriole.

We next analyzed LacZ staining in kidneys with recruited renin-expressing cells. Such an increase in the number of renin-expressing cells was induced by treating mice with a combination of a low-salt diet and an ACE inhibitor, as mentioned before. Clearly, also renin-expressing cells in these kidneys showed no or reduced Cx45-LacZ staining, but Cx40 immunoreactivity, while the surrounding vascular smooth muscle cells showed Cx45-LacZ staining but no Cx40 immunoreactivity (Figs. 5, D–F, and 6, C and D). Treatment of mice with a high-salt diet reduced the number of renin-expressing cells. Also under this condition, renin-expressing cells were negative for Cx45-LacZ staining but positive for Cx40 immunoreactivity (not shown).

DISCUSSION

This study aimed to determine the functional role of the gap junction protein Cx45 in renin-producing cells. In mice with a renin cell-specific deletion of the Cx45 gene, we did not find structural or functional abnormalities in renin-producing cells, as they were seen in mice lacking Cx40 protein in the renin cell lineage (23). In the absence of Cx40, renin-producing cells are located in the periglomerular interstitium instead in the media layer of afferent arterioles and these cells escape the control of renin secretion by renal perfusion pressure (11, 22). This defect leads to a relative or absolute hypersecretion of renin, which causes hypertension. Mice lacking Cx45 in renin-producing cells had apparently a normal renin system, and they were normotensive. We infer from these findings that Cx45 does not play a major functional role in the control of renin secretion. This conclusion is at first glance somewhat at odds with the results of a previous study reporting that mice lacking Cx45 in the nestin cell lineage had higher tissue and circulating renin levels and blood pressure values (7), suggesting a positive control function of Cx45 for renin synthesis and secretion and blood pressure. Since the nestin cell lineage comprises more cells (3, 14) than the renin cell lineage, including a wide distribution in the brain and adrenal glands, it cannot be excluded that the effects seen in mice with a disruption of the Cx45 gene in the nestin cell lineage were not related to indirect effects at the level of renin-producing cells. In that animal model, nestin-Cre/Cx45-flox recombination should lead to the expression of eGFP under the control of the Cx45 promoter. In fact, colocalization of eGFP fluorescence together with renin immunoreactivity was reported, suggesting that renin-producing cells express the Cx45 gene. Since we could not yet detect Cx45 in renin-producing cells by means of immunohistochemistry (10), we used another more direct strategy to test for the activity of the Cx45 gene promoter, namely, a LacZ reporter gene driven by the Cx45 promoter (9). In kidneys harboring the Cx45-LacZ gene, we found a strong expression of LacZ in the media layer of arteries and arterioles with the exception of renin-expressing cells. Cx45-LacZ staining in renin-expressing cells was much less if not absent in renin-expressing cells relative to the surrounding smooth muscle cells, indicating that promoter activity of the Cx45 gene is much lower in renin-expressing cells than in smooth muscle cells. Conversely, renin-producing cells expressed much higher levels of Cx40 than smooth muscle cells. Notably, this inverse pattern of Cx45 promoter activity and Cx40 expression in renin-producing and adjacent smooth muscle cells was a constant phenomenon independent of the degree of renin cell recruitment. We infer from this observation that the reversible metaplastic transformation of vascular smooth muscle cells into renin-producing cells and vice versa is accompanied by reciprocal changes in the transcriptional activities of the Cx40 and Cx45 genes. The (intra)cellular signaling cascades triggering the reversible phenotype switch of preglomerular vascular smooth muscle cells in the adult kidney are not understood. Apparently, the renin gene is not the only one that undergoes major transcriptional changes during this process. Finding more genes that are regulated in parallel or inversely with the renin gene could unravel intracellular master regulators of the phenotype switch.

GRANTS

This work was financially supported by the German Research Foundation (Grant WA 2137/2-1 and by SFB 699 to C. Wasgner and A. Kurtz and SFB 645 to K. Willecke). R. A. Gomez is supported by R37 HL066242 and RO1 HL096735 and M. L. Sequeira-Lopez by KO8 DK75481.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.