During nephrogenesis, renin expression shifts from large renal arteries toward smaller vessels in a defined spatiotemporal pattern, finally becoming restricted to the juxtaglomerular position. Chronic stimulation in adult kidneys leads to a recruitment of renin expression in the upstream vasculature. The mechanisms that control this characteristic switch-on and switch-off in the immature and adult kidney are not well-understood. Previous studies in mice with juxtaglomerular cell-specific deletion of the adenylyl cyclase-stimulatory G protein Gsα suggested that signaling along the cAMP pathway plays an essential role for renin expression during nephrogenesis and in the adult kidney. To identify the Gsα-dependent receptor that might be involved in activating this pathway, the present studies were performed to compare renin expression in wild types with that in mice with targeted deletions of β1 and β2-adrenoceptors. The sympathetic nervous system is an important regulator of the renin system in the adult kidney so that activation of β-adrenenoceptors may also participate in the activation of renin expression along the developing arterial tree and in upstream vasculature in adulthood. Compared with wild-types, renin expression was found to be significantly lower at all developmental stages in the kidneys of β1/β2 Adr−/− mice. Three-dimensional analysis showed reduced renin expression in all segments of the vascular tree in mutants and a virtual absence of renin expression in the large arcuate arteries. Adult mutant kidneys showed the typical upstream renin expression after chronic stimulation. Tyrosine hydroxylase staining in fetal and postnatal kidneys revealed that sympathetic innervation of renin-producing cells occurs early in fetal development. Our data indicate that genetic disruption of β-adrenergic receptors reduces basal renin expression along the developing preglomerular tree and in adult kidneys. Furthermore, β-adrenergic receptor input is critical for the expression of renin in large renal vessels during early fetal development.
- sympathetic nervous system
- renal development
- cAMP signaling
- ACE inhibition
early in the development of the mammalian kidney, renin-producing cells are located in the medial portion of the large renal arteries. As development progresses, renin expression shifts in a defined spatiotemporal pattern toward smaller arteries and afferent arterioles until it becomes restricted to its classic juxtaglomerular position at the vascular pole of the glomerulum. This characteristic process, which occurs in a similar pattern in all mammals (4, 9, 10, 13–15, 23, 24, 26, 30, 32, 34), suggests a systematic switch-on and switch-off of renin gene expression in cells integrated in the wall of the developing arteries and arterioles. Renin expression is plastic as well in adult kidneys. After chronic stimulation by classic maneuvers such as salt depletion, angiotensin-converting enzyme (ACE) inhibition, or renal artery stenosis, cells in the wall of afferent arterioles up to the interlobular arteries are stimulated to synthesize renin and the number of renin-producing cells is considerably increased (12).
The cAMP signaling pathway is an important stimulator of renin synthesis and secretion in the adult kidney. The role of the cAMP/PKA pathway in renin expression in adult kidneys has been definitively demonstrated by experiments in mice carrying a selective genetic deletion of the guanine nucleotide binding protein Gsα in juxtaglomerular cells (5). These mice have a very low basal level of renin expression that responds only marginally to classic stimuli (5). Furthermore, we showed that cAMP signaling is essential for the developmental changes in renin expression along the preglomerular vessel tree since interruption of cAMP signaling by Gsα deletion specifically in juxtaglomerular cells leads to a nearly complete abolition of embryonic renin expression in the kidney (31). It appears that the cAMP signaling pathway is critical for the initial appearance of renin in the larger renal arteries during kidney development and that it may also be required for the typical shift from large vessels to the classic juxtaglomerular position. The fact that Gsα mediates receptor-induced activation of adenylyl cyclase and cAMP formation implies that extracellular ligands that use the cAMP-dependent pathway are responsible for the characteristic spatiotemporal pattern of renin expression in the developing kidney or the upstream recruitment in adult mice. The identity of these ligands is unclear.
Catecholamines that signal through Gs-coupled β-adrenergic receptors on juxtaglomerular cells exert a clear effect on renin secretion and renin synthesis in the adult kidney (18). Activation of β-adrenoreceptors by catecholamines or β-adrenoreceptor agonists leads to an excess in cAMP production, which in turn markedly elevates the release and expression of renin (18). This stimulatory effect is believed to be mediated by postsynaptic β1-adrenoreceptors on the membrane of the juxtaglomerular granular cells and by presynaptic β2-adrenoreceptors, which are thought to amplify the synaptic distribution of norepinephrine (8). The presence of β1-adrenergic receptors in juxtaglomerular granular cells has been shown by receptor binding studies, in situ hybridization, and immunohistochemistry (3, 17, 29). Adult mice carrying deletions of both β1- and β2-adrenoreceptors have been shown to have substantially reduced basal renin expression and diminished regulatory responsiveness (20). Thus, β-adrenergic receptor activation by the sympathetic input from local nerve endings or from circulating catecholamines is thought to be significant for the maintenance of basal levels of renin synthesis and secretion.
In view of this major role of β-adrenergic receptors in the expression of renin and its regulation in the mature kidney, and because β-adrenergic receptors are expressed in fetal and mature kidneys (26), it is conceivable that β-adrenergic receptors play a role in determining renin expression during embryonic development and chronic stimulation. Therefore, the aims of this work were to characterize the spatiotemporal expression of renin in mouse kidneys developing in the absence of β1- and β2-adrenergic receptors and to study the sympathetic innervation of renin-producing cells in the immature kidney. Furthermore, we aimed to investigate the effects of β1 and β2 deficiency on the upstream recruitment of renin during stimulation of the renin system. Our results show that adrenergic innervation of renin-producing cells is present during fetal and postnatal life and remains in adulthood. We also found that the absence of β1- and β2-adrenergic receptors causes near absence of renin expression in large renal vessels during early embryonic development, whereas renin expression in afferent arterioles was reduced, but not markedly altered in its typical temporal pattern. In adult kidneys, renin expression during chronic stimulation by salt depletion and ACE blockade was not changed in mutant mice.
MATERIALS AND METHODS
β1/β2Adr−/− mice originally generated by Rohrer et al. (33) were obtained from Jackson Laboratories (Bar Harbor, ME) and were interbred to generate subsequent generations. The background of these animals contains genetic contributions from the FVB, C57Bl6, and 129SvJ strains. Wild-type (WT) animals were generated from the F2 generation of crosses between β1/β2Adr−/− and C57Bl6 mice. Four adult kidneys and three to five kidneys at each of the fetal [embryonic day 15 (E15), E16, E17, E18, E19] and postpartum (pp1, pp3, pp5, pp7, pp10, pp20) stages were examined from both strains. Furthermore, 8 to 10 adult mice of β1β2 knockout and WT strains were pretreated with a low-salt diet (0.02% sodium wt/wt) for 10 days. In addition, these mice received the ACE inhibitor enalapril (10 mg/kg) via the drinking water for the last 3 days of the dietary treatment. Genotyping was performed using standard protocols on DNA from the tail. Animal care and experimentation were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Determination of GAPDH and renin mRNA by real-time PCR.
Total RNA was isolated from frozen kidneys using the methods described by Chomczynski and Sacchi (7) and quantified using a photometer. One microgram of the resulting RNA was used for RT-PCR. The cDNA was synthesized by MMLV reverse transcriptase (Superscript, Invitrogen). For the quantification of renin mRNA expression (sense: 5′-ATG AAG GGG GTG TCT GTG GGG TC-3′, antisense: 5′-ATG CGG GGA GGG TGG GCA CCT G-3′), real-time RT-PCR was performed using a Light Cycler Instrument (Roche Diagnostics) and a QuantiTect SYBR Green PCR kit (Qiagen), with GAPDH (sense: 5′-TTC ATT GAC CTC AAC TAC AT-3′, antisense: 5′-GAG GGG CCA TCC ACA GTC TT-3′) as a control. PCR was run for 30 cycles with 15-s denaturation at 95°C, 20-s annealing at 60°C, and 20-s elongation at 72°C. The accuracy of the amplicon was verified by conducting a melting-curve analysis after amplification. All samples were run in duplicate.
Immunohistochemistry of renin, α-smooth muscle actin, and tyrosine hydroxylase.
After the death of the animals, kidneys from each of the E16 and E18 stage mice were removed and fixed in methyl Carnoy's solution (60% methanol, 30% chloroform, and 10% glacial acetic acid) at 4°C for 24 h. Kidneys from pp1 and adult mice were perfusion-fixed with 4% paraformaldehyde in PBS. The fixed kidneys were dehydrated in a graded series of alcohol solutions (2× in 70, 80, 90, and 100% methanol). Afterwards, each was further dehydrated using two 0.5-h incubations in 100% isopropanol and then embedded in paraffin.
Immunolabeling was performed on 5-μm sagittal paraffin sections. After being blocked with 10% horse serum/1% BSA in PBS for 0.5 h at room temperature, sections were incubated with chicken anti-renin IgG (diluted 1:400, Davids Biotechnologie), mouse anti-α-smooth muscle actin (α-SMA) IgG (diluted 1:100; Beckman Coulter, Immunotech), and rabbit anti-tyrosine hydroxylase (anti-TH) IgG (diluted 1:2,000; Millipore) overnight at 4°C. After several washing steps, the sections were incubated with Cy2-conjugated donkey anti-chicken IgG, Cy5-conjugated donkey anti-mouse IgG, and TRITC-conjugated donkey anti-rabbit IgG fluorescent antibodies (diluted 1:400, Dianova) for 1.5 h and were then mounted in glycergel (DakoCytomation).
Digitalization of the antibody-stained serial sections (up to 300 sections) was performed using an AxioCam MRm camera (Zeiss, Oberkochen, Germany) mounted on an Axiovert 200M microscope (Zeiss) with fluorescence filters for renin and α-SMA (TRITC, filter set 43; Cy2, filter set 38 HE; Zeiss). After acquisition, a stack of equal-size images was built using the graphic tool ImageJ (Wayne Rasband; NIH, Bethesda, MD). The equalized data were then imported into the Amira 4.1 visualization software (Mercury Computer Systems, Chelmsford, MA) on a Dell Precision 690 computer system (Dell) and split into the renin and α-SMA channels. After this step, the renin and α-SMA channels were aligned. In the segmentation step, the α-SMA and renin data sets served as a scaffold and were spanned manually or automatically using gray scale values. Matrices, volume surfaces, and statistics were generated from these segments.
Values are means ± SE. Differences between both genotypes were analyzed by ANOVA and Bonferroni's adjustment for multiple comparisons. Probability values <0.05 were considered statistically significant.
Developmental pattern of renin mRNA abundance.
To find out whether there are major differences in developmental renin gene expression between β-receptor-deficient mice and WT mice at all, we first determined relative renin mRNA levels in kidneys of both genotypes at different developmental stages (Fig. 1, top).
Renin mRNA abundance in WT kidneys increased during fetal kidney development, was highest around the date of birth, declined during the postnatal life, and reached lowest levels in the adult kidneys. Renin mRNA levels in β1/β2Adr−/− kidneys were lower than in WT at all developmental stages. The greatest differences were found during fetal development (Fig. 1, bottom), whereas the differences in renin mRNA levels between WT and β1/β2Adr−/− mice steadily decreased during postnatal life and became smallest in adults. (Fig. 1, top and bottom, and see Fig. 4).
Three-dimensional analysis of spatiotemporal developmental renin expression.
To assess the spatial aspects of developmental renin gene expression in β1/β2 Adr-deficient and WT mice, we determined the three-dimensional (3D) renin expression pattern in both strains of mice during kidney development.
Intrarenal renin expression sites and arterial preglomerular trees at different stages of development were reconstructed from serial sections stained with renin and α-SMA antibodies. Based on previous experience, the developmental stages E16, E18, pp1 were chosen, because at these time points major changes in renin expression occur in normal mice (31, 34). In WT mice, renin expression in the vessel wall first appeared early in fetal life, when renin-producing cells from an initial expression site at the large arcuate arteries nearly covered the entire surface of the developing renal arterial tree (E16; Fig. 2B). In contrast, β1/β2Adr−/− mice showed no renin expression in these vessel segments at that developmental stage and no divergence in the general shape of the vascular tree could be found in the kidneys of mice with β-adrenergic receptor deficiency (Fig. 2A). By day 18 of embryonic development, renin expression in WT mice had retreated to the more distal part of the preglomerular tree and started to expand in sprouting arcuate side arteries (Fig. 2D). In β1/β2Adr−/− mice, renin expression was present at this stage, but it was limited to the smaller vessels and was much less intense than in WT mice (Fig. 2C). On day 1 after birth, renin expression in WT mice had completely disappeared from arcuate arteries and was mainly found in vessel segments of the developing cortical vascular system (Fig. 3B). In β1/β2Adr−/− mice, renin-producing cells had a normal distribution, but there was explicitly less renin immunoreactivity in cortical vessel segments (Fig. 3A). We found again that the gross architecture of the preglomerular arterial vessel tree in β1/β2Adr−/− mice was not apparently different from that of WT at this stage and that structural and numeric abnormalities could not be demonstrated. Finally, in adult kidneys renin expression site was found at the classic juxtaglomerular position in both WT and β1/β2Adr−/− mice (Fig. 3, C and D).
Renin mRNA abundance and renin recruitment in adult kidneys.
In an analog approach to the ontogenetic considerations, we also investigated renin expression after stimulation by low salt and enalapril in kidneys of adult WT and β1/β2Adr−/− mice. Thereby, renin mRNA studies showed circa 20% lower basal renin mRNA levels in β1/β2Adr−/− mice than in WT (Fig. 4). After application of stimulation maneuvers, renin expression significantly increased in both genotypes. Although the increase of renin expression started at lower levels in mutant mice, it exactly gained the factor eight in WT and β1/β2Adr−/− knockouts (Fig. 4). To elucidate the recruitment of renin-producing cells in chronic stimulated kidneys, we performed a 3D reconstruction of the arteriolar vasculature and associated renin expression. With the use of this technique, no obvious differences between recruitment of renin-producing cells of WT and β1/β2Adr−/− knockouts were noted (Fig. 5).
Sympathetic innervation of renal blood vessels during kidney development.
The observation that β-adrenergic receptors might be particularly relevant for the early expression of renin in large vessels raised the question whether catecholamines as the ligands for β-adrenoreceptors can be found in the vicinity of renin-expressing cells. We therefore assessed the sympathetic innervation of renal blood vessels during fetal and postnatal development by immunohistochemical staining of the adrenergic innervation marker TH. At E16 when renin-producing cells were localized within the medial layer of the large renal arcuate arteries, sympathetic nerves were found along the sprouting arterial tree exhibiting a predominant perivascular distribution in the mouse kidney, and they also innervated the fetal renin-producing cells (Fig. 6A). On E18, the perivascular distribution of adrenergic nerve fibers was detected in close apposition to the renin-producing cells in the arcuate arcs and the renal side arteries (Fig. 6B). On day 1 after birth, renin expression was restricted to the distal part of the developing vessel tree and was found in the sprouting interlobular arteries and occasionally in the afferent arterioles, adrenergic fibers accompanied the branches of the maturing renal vessels and renin-producing cells appeared to have a rich supply of TH-positive adrenergic fibers (Fig. 6C). Staining of TH in adult kidneys clearly showed adrenergic nerve fibers in the immediate vicinity of the juxtaglomerular cells (Fig. 6D). To estimate the effects of β1β2 deletion for the sympathetic innervation of blood vessels and renin-producing cells in the kidney, we used TH immunohistochemistry as well in kidneys of β1/β2Adr−/− mice at different developmental stages. The outcome of this approach was that TH staining shows a similar pattern of adrenergic innervation in kidneys of fetal (Fig. 6E) and postnatal β1/β2Adr−/− mice (Fig. 6F) and reveals an unmodified distribution of adrenergic fibers in the adult mutant kidneys (data not shown).
The results of this study show that deletion of β1 and β2 adrenergic receptors is associated with a reduction of renin expression that is most pronounced in the developing mouse kidney when most of renin-expressing cells in WT mice are localized in the walls of the large renal arteries. This extensive renin expression along the arcuate arteries of embryonic kidneys was found to be nearly absent in the β-adrenergic receptor-deficient animals. In contrast, renin expression during early postnatal development was largely comparable between genotypes in its distribution and was seen in smaller vessels such as arcuate side arteries, sprouting interlobular arteries, and afferent arterioles. Thus, β-adrenergic receptors appear to be of critical importance for the initial appearance of renin in the larger vessels. Furthermore, presence of β-adrenergic receptors and presumably their activation do not appear to be required for the typical shift of renin expression from larger vessels to the juxtaglomerular portions of the afferent arterioles. Thus, initial appearance of renin expression in large vessels does not seem to be a prerequisite for the development of renin-producing cells in cortical small vessel segments. In addition to the differences in expression sites, renin expression levels in all locations were markedly lower in β1/β2Adr−/− than in WT mice, indicating that catecholamines affect basal renin expression in both embryonic and postnatal development.
Sympathetic nerves are known as determining stimulator for the renin system in adult kidneys. Accordingly the question arose whether renal nerves via β-adrenergic receptors can also influence the recruitment of renin-producing cells in the adult kidney. Application of chronic stimulation maneuvers reveals in mRNA measurements and 3D analysis that basal renin expression in mutant mice is lower than in WT (20), but stimulation remains unaffected and involves the elevation of the number of renin-producing cells. These data indicate that the recruitment of renin-producing cells in the adult kidney principally works without the influence of β-adrenergic receptors. The role of the sympathetic nervous system appears to be to act as cumulating factor for renin expression, but not as inductor in these recruited cells.
While it is generally accepted that renin-producing cells in the adult kidney are sympathetically innervated by β1-adrenergic receptors (3, 19, 22), the direct immunodemonstration of β1-receptors on renin-expressing cells has been hampered by the lack of suitable antibodies. Using TH expression as surrogate marker, our results show that the development of the intrarenal vascular tree is paralleled by perivascular sprouting of sympathetic nerves, thus providing a local source of catecholamines for the activation of β-adrenergic receptors. The development of sympathetic kidney innervations as observed in our study is in full accordance with previous reports in rats (1, 2), sheep (27), and dogs (11) as well as during human renal development (36). Specifically, our data indicate that sympathetic innervation of renin-producing cells is present during both fetal and early postnatal development. Similar results showing a strikingly early innervation of renin-producing cells have been obtained in a previous study in human fetuses in which TH-positive nerve fibers were detected in close apposition to the juxtaglomerular apparatus (36). Although TH is a well-documented marker of sympathetic nerve endings, its expression does not permit conclusions in regard to the identity of postsynaptic receptors. A clear differentiation between the different receptor types is not possible with currently available tools, and therefore direct evidence of the distribution of β1- and β2-adrenergic receptors during development cannot be provided at this time. Thus, we cannot confirm our suspicion that the markedly stronger effect of β-adrenoreceptor deletion on renin expression in large compared with smaller renal vessels may be a reflection of differential β-adrenoreceptor expression.
A comparison of developmental renin expression between β-adrenoreceptor-deficient mice with that in mice lacking the Gsα protein in renin-expressing cells indicates that β-receptor deficiency does not fully account for the phenotype of Gsα protein deficiency. Mice lacking the Gsα protein in renin-expressing cells fail to produce renin along the developing preglomerular vessel tree at any developmental stage (31). Because β1/β2-deficient mice do not completely mirror the renin phenotype of these mice, one has to assume that additional factors activate the cAMP signaling pathway and affect the development of renin expression. For example, cyclooxygenase-2 (Cox-2)-derived prostanoids exert profound effects on the release and synthesis of renin in the adult kidney (6, 16, 21, 28) by signaling via Gs-coupled prostacyclin (IP) and prostaglandin E (EP) receptors. However, previous studies showed that Cox-2 expression is very low during fetal and postnatal development and is highest at a time when the induction of nephrons and the maturation of the vascular system are largely complete (35). Dopamine signaling through Gs-coupled dopamine type 1a (Drd1a) receptors on juxtaglomerular cells is known to be an important positive regulator of renin synthesis and release in adult mice (25, 37). Dopamine is delivered via renal sympathetic nerves, but it can also come from intrarenal sources. Thus, it is conceivable that activation of Drd1a receptors during renal development is an additional factor that stimulates the cAMP/PKA cascade and influences the developmental renin expression. The effects of Cox-2 and Drd1a receptors on the developmental regulation of renin expression will be investigated in further experiments using the techniques presented in this study.
In contrast to the abnormal renin expression, the developing preglomerular arterial tree of β1/β2Adr−/− mice did not show obvious structural abnormalities in either immature or adult kidneys. Thus, signaling via β-adrenergic receptors does not play a major role in the development of the kidney vasculature.
Altogether, the results of our study suggest that cAMP signaling subsequent to β-receptor activation is critical for basal expression of vessel-associated renin during both kidney development and in adulthood. Perhaps more importantly, β-adrenergic receptors are required for renin expression in large renal vessels during fetal development, whereas condensation of renin expression in the juxtaglomerular position occurs in the absence of β-adrenergic input.
This study was financially supported by the Deutsche Forschungsgemeinschaft (SFB 699, WA 2-1).
No conflicts of interest, financial or otherwise, are declared by the author(s).
The expert technical assistance provided by Susanne Fink and Anna M'Bangui is gratefully acknowledged.