Homeostatic Responses in the Adrenal Cortex to the Absence of Aldosterone in Mice
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内分泌学杂志 2005年第6期
Departments of Pathology and Lab Medicine (G.L., N.M., K.C., O.S., H.-S.K.) and Cellular and Molecular Physiology (K.C.), University of North Carolina, Chapel Hill, North Carolina 27599-7525; Department of Pediatrics (M.L.S.L., R.A.G.), University of Virginia, Charlottesville, Virginia 22908; Department of Oral Biochemistry College of Dentistry (G.L.), Seoul National University, Seoul 110-749, South Korea; and Institute of Cytology and Genetics (N.M.), Siberian Division, Russian Academy of Sciences, Novosibirsk 630090, Russia
Address all correspondence and requests for reprints to: Hyung-Suk Kim, Ph.D., Department of Pathology and Laboratory Medicine, University of North Carolina, 703 Brinkhous-Bullitt Building, Chapel Hill, North Carolina 27599-7525. E-mail: hskim@med.unc.edu.
Abstract
To study the effects of decreased amounts or absence of aldosterone on development and endocrine function, we have disrupted the mouse gene, Cyp11b2, coding for aldosterone synthase (AS) by replacing its first two exons with sequences coding for enhanced green fluorescent protein. The null pups fail to thrive postnatally, and about 30% die between d 7 and 28. Aldosterone in plasma and AS mRNA in adrenal glands are undetectable in the null mice. Adult AS-null mice are small, weigh 75% of wild type, are hypotensive, have increased concentrations of plasma K+ and corticosterone, and a decreased concentration of plasma Cl–. Their plasma renin and angiotensin II concentrations are 45x and 4x wild type. The adrenal cortex is disorganized and has cells that contain marked accumulations of lipid. The zona glomerulosa is widened and includes easily detectable renin-containing cells, not seen in the wild-type adrenal gland. In the AS–/– adrenals, the level of mRNA for Cyp11b1, coding for 11?-hydroxylase, is 150% wild type. The adrenal glands of the null mice consequently show evidence of a greatly activated renin-angiotensin system and up-regulation of glucocorticoid production. In the AS-null mice enhanced green fluorescent protein fluorescence is mainly at the boundary between the cortex and medulla, where apoptotic cells are numerous. These data are consistent with the absence of aldosterone in the AS-null mice inducing an increased cell-turnover of cells in the adrenals that normally become AS expressing and their migration to the medullary boundary where they apoptose.
Introduction
THE RENIN-ANGIOTENSIN (Ang)-aldosterone system (RAAS) plays a key role in regulating blood pressure (BP) and salt balance. The RAAS regulates fluid and salt homeostasis through its effects on hemodynamics and vascular tone, on sodium reabsorption by the kidney, and on the secretion of aldosterone in adrenal gland (1, 2). Aldosterone is the most potent of the physiological mineralocorticoids that regulate Na+ and K+ homeostasis. Aldosterone consequently plays a direct role in maintaining electrolyte and water balance (3). Recently, it has been reported that aldosterone also plays a direct role in pathological remodeling of the heart, possibly by promoting fibrosis and cellular proliferation (4, 5).
Aldosterone is synthesized at the zona glomerulosa (zG) of adrenal cortex, which contains two other functional zones, the zona fasciculata (zF) and the zona reticularis (6, 7), from cholesterol by series of enzymic actions of which aldosterone synthase (AS; Cyp11b2) mediates the last two, 11?-hydroxylase and 18-hydroxylase and 18-methyl oxidase, using 11-deoxycorticosterone as its substrate (8, 9). The zG cells secrete aldosterone, regulated, by plasma Ang II and extracellular K+ concentration (10).
The main effect of aldosterone in the kidney is on transport of the electrolytes across epithelia, such as Na+ reabsorption and K+ secretion. This effect is mediated by the mineralocorticoid receptor (MR), which controls the activity of the epithelial Na channel in the luminal membrane of distal renal tubule cells (11, 12). A serum and glucocorticoid-induced serine/threonine kinase has recently been identified as an aldosterone-induced protein that activates epithelial Na channel (13). Glucocorticoids (cortisol in humans and corticosterone in rodents) also play an important role in the control of electrolytes (14). The mineralocorticoids aldosterone and glucocorticoid bind to intracellular MR and glucocorticoid receptor receptors with equal affinity. However, each of these receptors has a specific function that cannot be overcome by the other receptor as exemplified by the different phenotypes of the MR knockout (15) and glucocorticoid receptor knockout (16) mice. The cross-reaction between mineralocorticoid and glucocorticoid is minimized by various means, including differences in the localization of the two ligands and in their concentrations (circulating levels of GC exceed those of MC by about 1000-fold), and by the existence of 11?-hydroxysteroid dehydrogenase type 2, which converts GC to an inactive product (14, 17).
In adults, the juxtaglomerular cells are the primary source of plasma renin, a major homeostatic responder of the RAAS. The mouse renin gene is expressed mostly in the kidney, in the adrenal gland, and in the submandibular gland, but low levels of renin transcript are in the brain, heart, and other tissues (18). Expression of renin in the kidney and expression of AS in the adrenals are closely coupled and are influenced by Ang II (19).
The systemic effects and target tissues of aldosterone are well known, but there are still many unknowns in the control of adrenal steroid biogenesis and in the details of aldosterone action. To explore the pathophysiological effects of aldosterone deficiency, we therefore used homologous recombination in embryonic stem (ES) cells to generate mice in which the coding region of the AS (Cyp11b2) gene is disrupted by a cDNA coding for enhanced green fluorescent protein (EGFP). The resulting AS-null mice (AS–/–) are unable to synthesize aldosterone and show hypotension and high concentration of plasma K+, similar to humans lacking AS. In this paper, we present the physiological phenotypes of the AS-null mice and explore the responses of the adrenal glands of these mice to the absence of aldosterone.
Materials and Methods
Generation of AS-null mice
We cloned the AS gene from a strain 129/SvEv mouse genomic library. AS-null mice were generated with standard gene targeting methods as described previously (20). The targeting construct (Fig. 1B) included an EGFP coding region, in place of the coding sequence for AS, and a 3'-untranslated region from simian virus 40 polyA. The construct was electroporated into the TC-1 ES cell line (21) derived from mouse strain 129/SvEvTac; this targeting strategy leads to EGFP expression under the control of the AS promoter. After electroporation, G418/ganciclovir-resistant colonies were screened by PCR by using the primers depicted in Fig. 1C (5' TGG CGG ACC GCT ATC AGG AC 3' and 5' AAG CGG CCG CAA AGA TCC CTG AGA TAT T 3'). The targeted cells, which yield a 1.5-kb PCR product, were expanded, and their correctness was confirmed by Southern blot analysis with a HindIII/BamHI probe (Fig. 1, C and D). The targeted clones give a 9.0-kb band after SpeI digestion in addition to a 6.2-kb endogenous band (Fig. 1D).
FIG. 1. Generation of AS-null mice by targeted disruption of AS gene. A, Endogenous AS gene locus. The coding region is depicted by black box (B) Targeting construct. Neo, Neomycin resistance cassette; TK, thymidine kinase. C, Targeted allele after homologous recombination. The PCR primers are indicated by arrowheads and numbers, and the lengths of diagnostic restriction fragments and probe used for Southern analysis are shown. The PCR primers amplify a 1.5-kb fragment from the targeted allele. S, SpeI; H, HindIII; B, BamHI; X, XhoI; A, AscI; P, PacI; N, NotI. D, Detection of targeted allele by Southern analysis. Digestion of genomic DNA from targeted ES cells with SpeI results in a 6.2-bp fragment from endogenous allele and a 9.0-bp fragment from targeted allele when probed with the fragment depicted in C.
Male chimeras carrying the disrupted allele were mated with wild-type 129/SvEv females, and their male and female heterozygous progeny were mated to obtain AS-null mice with a genetically uniform background. To genotype the mice, DNA was isolated from a toe clip or from a small piece of tail tissue, followed by Southern blot analysis. Additionally, we determined the number of copies of the AS gene and Neo gene in the genomes of the mice using quantitative PCR (Applied Biosystems, Foster City, CA). All experiments were with 3- to 6-month-old male and female mice having the inbred strain 129 genetic background. The mice were handled following the National Institutes of Health guidelines for the use and care of experimental animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of North Carolina (Chapel Hill, NC).
BP measurements and analyses of blood
BP was measured in conscious mice using a computerized tail-cuff system (Visitech Systems, Cary, NC) as described (22).
Blood samples for measuring electrolytes were prepared by the following procedure. Mice were anesthetized by inhalation of 1.5–2.5% isoflurane in oxygen. To minimize hemolysis, blood samples were taken from the retro orbital sinus into 100-μl ammonium-heparinized capillary tubes and immediately centrifuged. The resulting plasma samples were analyzed within 1 h after bleeding with a VT250 Chemical Analyzer (Orthodiagnostic Clinical Inc., Rochester, NY) in the University of North Carolina Animal Clinical Laboratory Core Facility.
Blood samples for measuring plasma renin, Ang II, and steroid hormones were rapidly withdrawn from the descending aorta of mice after exposure to an atmosphere of CO2 (less than 1 min from loss of consciousness to the end of collection). This procedure reduces the effects of anesthesia on plasma renin activity (23, 24).
Plasma renin activity was measured as described (23) by RIA. RIAs for Ang II were performed as described previously (24), and for aldosterone (Diagnostic Products Corp., Los Angeles, CA) and corticosterone (ICN Biomedicals, Inc., Costa Mesa, CA) were performed using appropriate kits following the manufacturer’s protocols.
Histological and immunohistochemical analyses
For the histological analyses, 5-μm-thick paraffin sections of adrenal gland were stained with hematoxylin and eosin. Oil-Red-O staining was performed with frozen sections. Immunohistochemistry for renin in the adrenal gland sections (5-μm-thick paraffin sections) was performed with polyclonal renin antibody (1:10,000, gift from Dr. T. Inagami, Vanderbilt University, Nashville, TN) as described (25). Frozen tissue sections were used for adrenal gland EGFP detection. Apoptotic cells were detected with paraformaldehyde-fixed paraffin sections using the ApopTag Fluorescein In Situ Detection kit (Chemicon International, Temecula, CA), which is a modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Sections were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO).
Image analysis of Oil-Red-O staining
To quantitate levels of lipid accumulation, digitized images of Oil-Red-O-stained sections were analyzed with Scion Image 1.62a software (National Institutes of Health, Bethesda, MD). The intensities of staining in the red channel in the adrenal cortex were quantitated as the average of counts per pixel.
Quantitative RT-PCR for analysis of gene expression
Gene expression was measured by quantitative RT-PCR using the ABI Prism 7700 sequence detector (Applied Biosystems), as previously described (19). RNA was isolated from each tissue with the ABI Prism 6700 automated nucleic acid workstation following the manufacturer’s protocol. Primers and the corresponding fluorogenic probes for target genes were as shown in Table 1. Relative levels of gene expression as a percentage of wild type were determined for each gene. All assays were repeated twice, each with duplicates.
TABLE 1. TaqMan primers and probes
Statistics
Data analyses were performed with the JMP software (SAS Institute, Cary, NC). Values are presented as means ± SEM, and P values were calculated using Student’s t test for comparison of wild-type vs. AS–/– mice.
Results
Generation of AS-null mice
We disrupted the AS gene by homologous recombination in mouse ES cells. As shown in Fig. 1, exons 1, 2, part of exon 3, and introns 1 and 2 of the AS gene are replaced by sequences coding for EGFP and the Neo gene. The disrupted allele was identified in ES cells by PCR and confirmed by Southern blot analysis as shown in Fig. 1D. The targeted ES cells were injected into blastocyst, and male chimeras were mated to 129SvEv females. Resulting male and female AS+/– heterozygotes were mated. Their progeny included AS+/+, +/–, and –/– pups in the expected Mendelian ratios at birth. The AS–/– pups, however, failed to thrive, and approximately 30% of them died between 7 and 28 d (69 +/+, 134+/–, and 48 –/– at 28 d). To test whether the targeting had inactivated the AS gene in the AS-null mice, quantitative RT-PCR was performed on total RNA from the adrenal gland. Expression of AS mRNA in adrenal gland was undetectable in the AS–/– mice, confirming complete loss of AS production (see Table 3).
TABLE 3. mRNA levels in the adrenal glands measured by quantitative RT PCR
Changes of body and organ weights in AS–/– mice
Adult AS–/– mice are small and weigh about 75% of their wild-type littermates. The weights of the heart, kidney, and liver of the AS–/– mice are significantly less than wild type, but these differences are not significant after normalizing for body weight (Table 2).
TABLE 2. Body and organ weights and blood data in AS+/+ and AS–/– mice
Main physiological phenotypes of AS–/– mice
Table 2 shows that aldosterone was undetectable in the plasma of the AS–/– mice. The plasma concentration of corticosterone was significantly increased in the AS–/– mice (79.0 ± 11.0 vs. wild-type, 51.2 ± 11.1 ng/ml; P < 0.05). The concentration of K+ in plasma was significantly increased in the AS–/– mice (6.1 ± 0.1 vs. wild-type, 5.2 ± 0.1 mM; P < 0.05). The concentration of Cl– in plasma was significantly decreased in the AS–/– mice (128.8 ± 0.6 vs. wild-type, 133.1 ± 0.6 mM; P < 0.05), but the concentration of Na+ was not statistically different from wild type.
We used a computerized tail-cuff system to measure BP, and Fig. 2A shows that the AS–/– mice have significantly lower BP than wild type (111.7 vs. 97.3 mm Hg; P < 0.01), indicating a mild degree of hypotension. Figure 2, B and C, show the plasma levels of renin and Ang II in the AS+/+ and AS–/– mice. Plasma renin was more than 45 times higher in the AS–/– mice than in the AS+/+ mice (28 vs. 1300 ng Ang I/ml·h; P < 0.001). The plasma concentration of Ang II was increased about 4-fold in the AS–/– mice (59 vs. 229 pg/ml; P < 0.0001). The AS–/– mice are consequently unique in being hypotensive but having very high levels of plasma renin and high levels of Ang II.
FIG. 2. BP and plasma renin and Ang II levels in wild-type (+/+) and AS-null (–/–) mice. A, BP (millimeters of Hg); B, plasma renin concentration (nanograms of Ang I per milliliter per hour); C, plasma Ang II concentration (picograms per milliliter). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 for +/+ vs. –/–.
Changes in the adrenal gland
Histological examination of the adrenal glands in the AS–/– mice showed that the cortex was disorganized and that the glomerulosa was thickened. In paraffin sections, the cells in the adrenal cortex appeared foamy and had a vacuolated cytoplasm and abnormal shapes (Fig. 3, A–D). Frozen sections stained with Oil-Red-O showed that the vacuoles reflect an accumulation of lipid in the steroidogenic adrenal cortex (Fig. 3, E and F). Using image analysis of digitized forms of the pictures shown in Fig. 3, E and F, the average intensities of Oil-Red-O staining over the cortex were 1040 counts/pixel in wild-type mice and 3600 counts/pixel in AS–/– mice, indicating approximately 3 times more lipid in the adrenal cortex of AS–/– mice.
FIG. 3. Histological analysis of the adrenal gland of wild-type (+/+) and AS-null (–/–) mice. Hematoxylin and eosin staining micrographs of adrenal gland of wild-type (A) and AS-null (B) mice at low magnification (x20) and at higher magnification (x40) with wild-type (C) and –/– (D) mice. Oil-Red-O staining shows localization of lipid in wild-type (E) and AS–/– mice (F) with x20 magnification.
Localization of the renin-producing cells in the adrenal glands
Although renin is produced in the zG of adult wild-type mice, it is difficult to detect immunochemically because of its low level, as shown in Fig. 4A. However, in the AS–/– mice, many renin-staining cells are apparent in the zG and even in some parts of the zF (Fig. 4B).
FIG. 4. Immunohistochemistry for renin. The cortex of adrenal glands of wild-type (A) and AS–/– (B) mice are shown at high magnifications (x400) with the capsule of the adrenal gland toward the lower left.
Characterization of the adrenal cells in AS–/– mice
Because the AS-null allele was generated by replacing the coding region of the natural gene with sequences coding for EGFP, we could determine where the modified gene is expressed. Surprisingly, we found the strongest fluorescent signals at the boundary between the cortex and medulla of the adrenal gland in the AS–/– mice (Fig. 5B). No fluorescent signals were detectable at this boundary in the adrenals of wild-type mice (Fig. 5A) or in the heterozygotes (data not shown). Because the cortex/medulla boundary area accumulates dead cells after migration of damaged cells from the cortex (26), we used the TUNEL assay to look for apoptotic cells as shown in Fig. 5, C and D. Figure 5D shows a clear signal mainly at the boundary area between cortex and medulla in the AS–/– mice. Faint signals are detectable in the zF. No signals are detected in the wild type with the same staining procedure (Fig. 5C).
FIG. 5. Cellular detection assays in the adrenal glands of wild-type (+/+) and AS-null mice (–/–). EGFP signals in wild-type (A) and AS–/– (B) mice at x200 magnification. TUNEL assay signals in wild-type (C) and AS–/– (D) mice. The nuclei, stained with Hoechst 33258, appear blue; the TUNEL-positive areas are pseudo-colored yellow.
RNA studies in the adrenal gland
To look for any changes in the pathway for adrenal steroid hormone synthesis in AS–/– mice, we analyzed the mRNA levels of several relevant genes by quantitative RT-PCR as shown in Table 3. The results show that expression of the AS gene (Cyp11b2) was not detectable in the AS–/– mice. The mRNA level in the heterozygous mice (AS+/–) was 65% of wild-type level, but this was not significantly different from the expected level of 50%. However, in the AS–/– mice, the level of EGFP mRNA (a surrogate for the natural gene) was 7 times more than the level in the AS+/– mice, indicating a very substantial homeostatic increase in activity of the gene in the null mice. In the zF of the adrenal gland of the AS–/– mice, expression of Cyp11b1 mRNA, which codes for 11?-hydroxylase, was increased to about 150% of that in wild-type mice. Expression in the AS–/– adrenals of the gene for Cyp21A, which produces 11-deoxycorticosterone from progesterone, was 62% of that in wild-type mice. Expression of the gene for the steroidogenic acute regulatory protein (StAR), which is essential for translocation of cholesterol from cytoplasm to the inner mitochondrial membrane, was also decreased, to 56% of the level in wild-type mice. Low-density lipoprotein receptor, which is important for the transport of cholesterol into the adrenal cells, was unchanged in expression.
As expected from the immunochemical results, renin mRNA in the adrenals of the AS–/– male and female mice was very markedly increased, to 100 times wild-type mice. Expression of the Atr1b gene, which is the main Ang II receptor in the zG (27), was increased 7 times relative to wild type, whereas expression of the Atr1a gene, which is the Ang II receptor in the zF and zG (28), was only slightly increased.
Although, as indicated above, the heterozygous mice (AS+/–) have AS mRNA levels reduced to about half of wild-type, they have no morphological or physiological abnormalities and show no differences in the levels of expression of the genes that we tested in the AS–/– animals.
Discussion
We have generated and characterized AS-null mice (AS–/–) and have shown that they are unable to synthesize aldosterone. About 70% of the AS-null mice survive to weaning but have decreased body size, failure to thrive, hypotension, increased concentrations of plasma K+ and corticosterone, a decreased concentration of plasma Cl–, and very high plasma renin activity.
There are two well-known AS deficiencies in humans: corticosterone methyloxidase deficiency types I and II (29). Type I is caused by mutations in CYP11B2 that result in the expression of AS that has lost all of its activities. The type I patients have undetectable aldosterone levels, increased levels of 18-hydroxy-11-deoxycorticosterone, and reduced levels of 18-hydroxycorticosterone. The patients have severe salt loss, hypotension, hyperkalemia, hyponatremia, and very high plasma renin activity. They also have metabolic acidosis as neonates, fail to thrive in infancy, and show growth retardation in early childhood (30, 31). The type II form of AS deficiency in humans is characterized by low aldosterone and increased 18-hydroxycorticosterone levels. It is caused by mutations that impair the 18-hydroxylase and 18 methyl-oxidase activities of AS, but the 11?-hydroxylase activity is retained. These patients have a phenotype closely similar to the type I phenotype.
The AS-null mice share all the features of the type I human patients, including a very high plasma renin activity (45x wild-type level). Their plasma Ang II levels are increased to about 4x wild-type. Yet, the mice are hypotensive (BP about 14 mm Hg below wild type). Thus, the AS–/– mice, like type I corticosterone methyloxidase-deficient humans and MR-deficient mice (15), are unable to maintain a normal BP in the absence of aldosterone, despite their high plasma renin and increased Ang II levels. Nevertheless, it is clear that compensatory mechanisms that strongly activate the renin-Ang system (RAS) and up-regulate glucocorticoid production have been induced in the AS–/– mice. However, these compensations are not sufficient to normalize the plasma K+ and Cl– concentrations altered by the absence of aldosterone.
Comment is required on the survival of about 70% of our AS–/– pups and the absence of hyponatremia and salt wasting without salt supplementation compared with the absence of survival of either adrenalectomized animals or MR–/– mice (15) without supplementation. A possible explanation of this difference is that the AS–/– mice retain some control of sodium transport via up-regulated production of glucocorticoids that can act through the MR (32, 33, 34). This contrasts with the adrenalectomized animals, which are more handicapped because they cannot synthesize ligand, mineralocorticoid, or glucocorticoid, and the MR–/– mice, which lack the receptor.
Our data show that absence of aldosterone induces marked cellular changes in the adrenal cortex. The most easily detected response in the AS–/– mice is a massive increase in renin gene expression (100x wild-type level) in the adrenal cortex. This response is in accordance with previous work showing that in the adrenal cortex, the RAS plays a key role in controlling the secretion of aldosterone (10). Our data suggest that the easy detection of renin-containing cells in the adrenals of the AS–/– mice results from an increase in the amount of renin produced in individual renin producing cells possibly combined with an increase in their numbers. [Increases in the number of renin-containing cells are demonstrated in the kidneys of the aldosterone-deficient mice (Makhanova, M., G. Lee, M. L. Sequeira Lopez, R. A. Gomez, H.-S. Kim, N. Takahashi, and O. Smithies, unpublished data) and in mice lacking Ang (24), Ang-converting enzyme (35), or Atr1a (36).] Another indication of greatly increased RAS activity in the adrenals of the AS–/– mice is a 7-fold increase in the expression of Atr1b, the most abundant receptor of Ang II in the zG.
The absence of aldosterone in the AS–/– mice leads to several abnormalities in adrenal structure, including widening of the glomerulosa, an abnormal morphology of the fasciculata cells that have foamy and vacuolated cytoplasms caused by a greatly increased accumulation of lipid. This lipid accumulation is very similar to the adrenal lipid hyperplasia caused by absence of StAR, the StAR (37), or of Cyp11A1, the cholesterol side-chain cleavage enzyme (38). Interestingly, the mRNA levels of StAR and Cyp21A in the AS–/– mice are reduced to approximately 50% wild type, indicating down-regulation of the steroidogenic pathways.
Several features observed in the adrenals of the AS–/– mice indicate an increase in cell turnover. Thus, we found that the strongest EGFP signal, a marker for AS gene expression, was at the morphological boundary between the cortex and medulla, but these cells were highly apoptotic. Taken together, our data strongly support the hypothesis that increased RAS activity in the adrenal cortex induces the zG cells to undergo more cell turnover, presumably in an attempt to produce more aldosterone, and that these cells migrate through the cortex and enter apoptosis at the boundary between cortex and medulla.
In summary, we describe here a cellular and molecular characterization of aldosterone deficiency in mice and of the homeostatic changes induced in their adrenal glands. Despite a massive increase in plasma renin and Ang II, the mice are still hypotensive. The cortex of their adrenals shows a remarkable increase in the migration and apoptosis of cells that normally produce aldosterone. Renin-producing cells are easily demonstrated in the adrenal cortex in association with a 100-fold increase in adrenal renin mRNA. Therefore, the AS–/– mice should be valuable for studying how aldosterone regulates its own synthesis in the adrenals and affects the synthesis of renin in the adrenal glands and elsewhere.
Acknowledgments
We thank Kimberly Kluckman, Emily Riggs, and Gang Cui for technical assistance.
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Address all correspondence and requests for reprints to: Hyung-Suk Kim, Ph.D., Department of Pathology and Laboratory Medicine, University of North Carolina, 703 Brinkhous-Bullitt Building, Chapel Hill, North Carolina 27599-7525. E-mail: hskim@med.unc.edu.
Abstract
To study the effects of decreased amounts or absence of aldosterone on development and endocrine function, we have disrupted the mouse gene, Cyp11b2, coding for aldosterone synthase (AS) by replacing its first two exons with sequences coding for enhanced green fluorescent protein. The null pups fail to thrive postnatally, and about 30% die between d 7 and 28. Aldosterone in plasma and AS mRNA in adrenal glands are undetectable in the null mice. Adult AS-null mice are small, weigh 75% of wild type, are hypotensive, have increased concentrations of plasma K+ and corticosterone, and a decreased concentration of plasma Cl–. Their plasma renin and angiotensin II concentrations are 45x and 4x wild type. The adrenal cortex is disorganized and has cells that contain marked accumulations of lipid. The zona glomerulosa is widened and includes easily detectable renin-containing cells, not seen in the wild-type adrenal gland. In the AS–/– adrenals, the level of mRNA for Cyp11b1, coding for 11?-hydroxylase, is 150% wild type. The adrenal glands of the null mice consequently show evidence of a greatly activated renin-angiotensin system and up-regulation of glucocorticoid production. In the AS-null mice enhanced green fluorescent protein fluorescence is mainly at the boundary between the cortex and medulla, where apoptotic cells are numerous. These data are consistent with the absence of aldosterone in the AS-null mice inducing an increased cell-turnover of cells in the adrenals that normally become AS expressing and their migration to the medullary boundary where they apoptose.
Introduction
THE RENIN-ANGIOTENSIN (Ang)-aldosterone system (RAAS) plays a key role in regulating blood pressure (BP) and salt balance. The RAAS regulates fluid and salt homeostasis through its effects on hemodynamics and vascular tone, on sodium reabsorption by the kidney, and on the secretion of aldosterone in adrenal gland (1, 2). Aldosterone is the most potent of the physiological mineralocorticoids that regulate Na+ and K+ homeostasis. Aldosterone consequently plays a direct role in maintaining electrolyte and water balance (3). Recently, it has been reported that aldosterone also plays a direct role in pathological remodeling of the heart, possibly by promoting fibrosis and cellular proliferation (4, 5).
Aldosterone is synthesized at the zona glomerulosa (zG) of adrenal cortex, which contains two other functional zones, the zona fasciculata (zF) and the zona reticularis (6, 7), from cholesterol by series of enzymic actions of which aldosterone synthase (AS; Cyp11b2) mediates the last two, 11?-hydroxylase and 18-hydroxylase and 18-methyl oxidase, using 11-deoxycorticosterone as its substrate (8, 9). The zG cells secrete aldosterone, regulated, by plasma Ang II and extracellular K+ concentration (10).
The main effect of aldosterone in the kidney is on transport of the electrolytes across epithelia, such as Na+ reabsorption and K+ secretion. This effect is mediated by the mineralocorticoid receptor (MR), which controls the activity of the epithelial Na channel in the luminal membrane of distal renal tubule cells (11, 12). A serum and glucocorticoid-induced serine/threonine kinase has recently been identified as an aldosterone-induced protein that activates epithelial Na channel (13). Glucocorticoids (cortisol in humans and corticosterone in rodents) also play an important role in the control of electrolytes (14). The mineralocorticoids aldosterone and glucocorticoid bind to intracellular MR and glucocorticoid receptor receptors with equal affinity. However, each of these receptors has a specific function that cannot be overcome by the other receptor as exemplified by the different phenotypes of the MR knockout (15) and glucocorticoid receptor knockout (16) mice. The cross-reaction between mineralocorticoid and glucocorticoid is minimized by various means, including differences in the localization of the two ligands and in their concentrations (circulating levels of GC exceed those of MC by about 1000-fold), and by the existence of 11?-hydroxysteroid dehydrogenase type 2, which converts GC to an inactive product (14, 17).
In adults, the juxtaglomerular cells are the primary source of plasma renin, a major homeostatic responder of the RAAS. The mouse renin gene is expressed mostly in the kidney, in the adrenal gland, and in the submandibular gland, but low levels of renin transcript are in the brain, heart, and other tissues (18). Expression of renin in the kidney and expression of AS in the adrenals are closely coupled and are influenced by Ang II (19).
The systemic effects and target tissues of aldosterone are well known, but there are still many unknowns in the control of adrenal steroid biogenesis and in the details of aldosterone action. To explore the pathophysiological effects of aldosterone deficiency, we therefore used homologous recombination in embryonic stem (ES) cells to generate mice in which the coding region of the AS (Cyp11b2) gene is disrupted by a cDNA coding for enhanced green fluorescent protein (EGFP). The resulting AS-null mice (AS–/–) are unable to synthesize aldosterone and show hypotension and high concentration of plasma K+, similar to humans lacking AS. In this paper, we present the physiological phenotypes of the AS-null mice and explore the responses of the adrenal glands of these mice to the absence of aldosterone.
Materials and Methods
Generation of AS-null mice
We cloned the AS gene from a strain 129/SvEv mouse genomic library. AS-null mice were generated with standard gene targeting methods as described previously (20). The targeting construct (Fig. 1B) included an EGFP coding region, in place of the coding sequence for AS, and a 3'-untranslated region from simian virus 40 polyA. The construct was electroporated into the TC-1 ES cell line (21) derived from mouse strain 129/SvEvTac; this targeting strategy leads to EGFP expression under the control of the AS promoter. After electroporation, G418/ganciclovir-resistant colonies were screened by PCR by using the primers depicted in Fig. 1C (5' TGG CGG ACC GCT ATC AGG AC 3' and 5' AAG CGG CCG CAA AGA TCC CTG AGA TAT T 3'). The targeted cells, which yield a 1.5-kb PCR product, were expanded, and their correctness was confirmed by Southern blot analysis with a HindIII/BamHI probe (Fig. 1, C and D). The targeted clones give a 9.0-kb band after SpeI digestion in addition to a 6.2-kb endogenous band (Fig. 1D).
FIG. 1. Generation of AS-null mice by targeted disruption of AS gene. A, Endogenous AS gene locus. The coding region is depicted by black box (B) Targeting construct. Neo, Neomycin resistance cassette; TK, thymidine kinase. C, Targeted allele after homologous recombination. The PCR primers are indicated by arrowheads and numbers, and the lengths of diagnostic restriction fragments and probe used for Southern analysis are shown. The PCR primers amplify a 1.5-kb fragment from the targeted allele. S, SpeI; H, HindIII; B, BamHI; X, XhoI; A, AscI; P, PacI; N, NotI. D, Detection of targeted allele by Southern analysis. Digestion of genomic DNA from targeted ES cells with SpeI results in a 6.2-bp fragment from endogenous allele and a 9.0-bp fragment from targeted allele when probed with the fragment depicted in C.
Male chimeras carrying the disrupted allele were mated with wild-type 129/SvEv females, and their male and female heterozygous progeny were mated to obtain AS-null mice with a genetically uniform background. To genotype the mice, DNA was isolated from a toe clip or from a small piece of tail tissue, followed by Southern blot analysis. Additionally, we determined the number of copies of the AS gene and Neo gene in the genomes of the mice using quantitative PCR (Applied Biosystems, Foster City, CA). All experiments were with 3- to 6-month-old male and female mice having the inbred strain 129 genetic background. The mice were handled following the National Institutes of Health guidelines for the use and care of experimental animals. All experiments were approved by the Institutional Animal Care and Use Committee of the University of North Carolina (Chapel Hill, NC).
BP measurements and analyses of blood
BP was measured in conscious mice using a computerized tail-cuff system (Visitech Systems, Cary, NC) as described (22).
Blood samples for measuring electrolytes were prepared by the following procedure. Mice were anesthetized by inhalation of 1.5–2.5% isoflurane in oxygen. To minimize hemolysis, blood samples were taken from the retro orbital sinus into 100-μl ammonium-heparinized capillary tubes and immediately centrifuged. The resulting plasma samples were analyzed within 1 h after bleeding with a VT250 Chemical Analyzer (Orthodiagnostic Clinical Inc., Rochester, NY) in the University of North Carolina Animal Clinical Laboratory Core Facility.
Blood samples for measuring plasma renin, Ang II, and steroid hormones were rapidly withdrawn from the descending aorta of mice after exposure to an atmosphere of CO2 (less than 1 min from loss of consciousness to the end of collection). This procedure reduces the effects of anesthesia on plasma renin activity (23, 24).
Plasma renin activity was measured as described (23) by RIA. RIAs for Ang II were performed as described previously (24), and for aldosterone (Diagnostic Products Corp., Los Angeles, CA) and corticosterone (ICN Biomedicals, Inc., Costa Mesa, CA) were performed using appropriate kits following the manufacturer’s protocols.
Histological and immunohistochemical analyses
For the histological analyses, 5-μm-thick paraffin sections of adrenal gland were stained with hematoxylin and eosin. Oil-Red-O staining was performed with frozen sections. Immunohistochemistry for renin in the adrenal gland sections (5-μm-thick paraffin sections) was performed with polyclonal renin antibody (1:10,000, gift from Dr. T. Inagami, Vanderbilt University, Nashville, TN) as described (25). Frozen tissue sections were used for adrenal gland EGFP detection. Apoptotic cells were detected with paraformaldehyde-fixed paraffin sections using the ApopTag Fluorescein In Situ Detection kit (Chemicon International, Temecula, CA), which is a modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Sections were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO).
Image analysis of Oil-Red-O staining
To quantitate levels of lipid accumulation, digitized images of Oil-Red-O-stained sections were analyzed with Scion Image 1.62a software (National Institutes of Health, Bethesda, MD). The intensities of staining in the red channel in the adrenal cortex were quantitated as the average of counts per pixel.
Quantitative RT-PCR for analysis of gene expression
Gene expression was measured by quantitative RT-PCR using the ABI Prism 7700 sequence detector (Applied Biosystems), as previously described (19). RNA was isolated from each tissue with the ABI Prism 6700 automated nucleic acid workstation following the manufacturer’s protocol. Primers and the corresponding fluorogenic probes for target genes were as shown in Table 1. Relative levels of gene expression as a percentage of wild type were determined for each gene. All assays were repeated twice, each with duplicates.
TABLE 1. TaqMan primers and probes
Statistics
Data analyses were performed with the JMP software (SAS Institute, Cary, NC). Values are presented as means ± SEM, and P values were calculated using Student’s t test for comparison of wild-type vs. AS–/– mice.
Results
Generation of AS-null mice
We disrupted the AS gene by homologous recombination in mouse ES cells. As shown in Fig. 1, exons 1, 2, part of exon 3, and introns 1 and 2 of the AS gene are replaced by sequences coding for EGFP and the Neo gene. The disrupted allele was identified in ES cells by PCR and confirmed by Southern blot analysis as shown in Fig. 1D. The targeted ES cells were injected into blastocyst, and male chimeras were mated to 129SvEv females. Resulting male and female AS+/– heterozygotes were mated. Their progeny included AS+/+, +/–, and –/– pups in the expected Mendelian ratios at birth. The AS–/– pups, however, failed to thrive, and approximately 30% of them died between 7 and 28 d (69 +/+, 134+/–, and 48 –/– at 28 d). To test whether the targeting had inactivated the AS gene in the AS-null mice, quantitative RT-PCR was performed on total RNA from the adrenal gland. Expression of AS mRNA in adrenal gland was undetectable in the AS–/– mice, confirming complete loss of AS production (see Table 3).
TABLE 3. mRNA levels in the adrenal glands measured by quantitative RT PCR
Changes of body and organ weights in AS–/– mice
Adult AS–/– mice are small and weigh about 75% of their wild-type littermates. The weights of the heart, kidney, and liver of the AS–/– mice are significantly less than wild type, but these differences are not significant after normalizing for body weight (Table 2).
TABLE 2. Body and organ weights and blood data in AS+/+ and AS–/– mice
Main physiological phenotypes of AS–/– mice
Table 2 shows that aldosterone was undetectable in the plasma of the AS–/– mice. The plasma concentration of corticosterone was significantly increased in the AS–/– mice (79.0 ± 11.0 vs. wild-type, 51.2 ± 11.1 ng/ml; P < 0.05). The concentration of K+ in plasma was significantly increased in the AS–/– mice (6.1 ± 0.1 vs. wild-type, 5.2 ± 0.1 mM; P < 0.05). The concentration of Cl– in plasma was significantly decreased in the AS–/– mice (128.8 ± 0.6 vs. wild-type, 133.1 ± 0.6 mM; P < 0.05), but the concentration of Na+ was not statistically different from wild type.
We used a computerized tail-cuff system to measure BP, and Fig. 2A shows that the AS–/– mice have significantly lower BP than wild type (111.7 vs. 97.3 mm Hg; P < 0.01), indicating a mild degree of hypotension. Figure 2, B and C, show the plasma levels of renin and Ang II in the AS+/+ and AS–/– mice. Plasma renin was more than 45 times higher in the AS–/– mice than in the AS+/+ mice (28 vs. 1300 ng Ang I/ml·h; P < 0.001). The plasma concentration of Ang II was increased about 4-fold in the AS–/– mice (59 vs. 229 pg/ml; P < 0.0001). The AS–/– mice are consequently unique in being hypotensive but having very high levels of plasma renin and high levels of Ang II.
FIG. 2. BP and plasma renin and Ang II levels in wild-type (+/+) and AS-null (–/–) mice. A, BP (millimeters of Hg); B, plasma renin concentration (nanograms of Ang I per milliliter per hour); C, plasma Ang II concentration (picograms per milliliter). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 for +/+ vs. –/–.
Changes in the adrenal gland
Histological examination of the adrenal glands in the AS–/– mice showed that the cortex was disorganized and that the glomerulosa was thickened. In paraffin sections, the cells in the adrenal cortex appeared foamy and had a vacuolated cytoplasm and abnormal shapes (Fig. 3, A–D). Frozen sections stained with Oil-Red-O showed that the vacuoles reflect an accumulation of lipid in the steroidogenic adrenal cortex (Fig. 3, E and F). Using image analysis of digitized forms of the pictures shown in Fig. 3, E and F, the average intensities of Oil-Red-O staining over the cortex were 1040 counts/pixel in wild-type mice and 3600 counts/pixel in AS–/– mice, indicating approximately 3 times more lipid in the adrenal cortex of AS–/– mice.
FIG. 3. Histological analysis of the adrenal gland of wild-type (+/+) and AS-null (–/–) mice. Hematoxylin and eosin staining micrographs of adrenal gland of wild-type (A) and AS-null (B) mice at low magnification (x20) and at higher magnification (x40) with wild-type (C) and –/– (D) mice. Oil-Red-O staining shows localization of lipid in wild-type (E) and AS–/– mice (F) with x20 magnification.
Localization of the renin-producing cells in the adrenal glands
Although renin is produced in the zG of adult wild-type mice, it is difficult to detect immunochemically because of its low level, as shown in Fig. 4A. However, in the AS–/– mice, many renin-staining cells are apparent in the zG and even in some parts of the zF (Fig. 4B).
FIG. 4. Immunohistochemistry for renin. The cortex of adrenal glands of wild-type (A) and AS–/– (B) mice are shown at high magnifications (x400) with the capsule of the adrenal gland toward the lower left.
Characterization of the adrenal cells in AS–/– mice
Because the AS-null allele was generated by replacing the coding region of the natural gene with sequences coding for EGFP, we could determine where the modified gene is expressed. Surprisingly, we found the strongest fluorescent signals at the boundary between the cortex and medulla of the adrenal gland in the AS–/– mice (Fig. 5B). No fluorescent signals were detectable at this boundary in the adrenals of wild-type mice (Fig. 5A) or in the heterozygotes (data not shown). Because the cortex/medulla boundary area accumulates dead cells after migration of damaged cells from the cortex (26), we used the TUNEL assay to look for apoptotic cells as shown in Fig. 5, C and D. Figure 5D shows a clear signal mainly at the boundary area between cortex and medulla in the AS–/– mice. Faint signals are detectable in the zF. No signals are detected in the wild type with the same staining procedure (Fig. 5C).
FIG. 5. Cellular detection assays in the adrenal glands of wild-type (+/+) and AS-null mice (–/–). EGFP signals in wild-type (A) and AS–/– (B) mice at x200 magnification. TUNEL assay signals in wild-type (C) and AS–/– (D) mice. The nuclei, stained with Hoechst 33258, appear blue; the TUNEL-positive areas are pseudo-colored yellow.
RNA studies in the adrenal gland
To look for any changes in the pathway for adrenal steroid hormone synthesis in AS–/– mice, we analyzed the mRNA levels of several relevant genes by quantitative RT-PCR as shown in Table 3. The results show that expression of the AS gene (Cyp11b2) was not detectable in the AS–/– mice. The mRNA level in the heterozygous mice (AS+/–) was 65% of wild-type level, but this was not significantly different from the expected level of 50%. However, in the AS–/– mice, the level of EGFP mRNA (a surrogate for the natural gene) was 7 times more than the level in the AS+/– mice, indicating a very substantial homeostatic increase in activity of the gene in the null mice. In the zF of the adrenal gland of the AS–/– mice, expression of Cyp11b1 mRNA, which codes for 11?-hydroxylase, was increased to about 150% of that in wild-type mice. Expression in the AS–/– adrenals of the gene for Cyp21A, which produces 11-deoxycorticosterone from progesterone, was 62% of that in wild-type mice. Expression of the gene for the steroidogenic acute regulatory protein (StAR), which is essential for translocation of cholesterol from cytoplasm to the inner mitochondrial membrane, was also decreased, to 56% of the level in wild-type mice. Low-density lipoprotein receptor, which is important for the transport of cholesterol into the adrenal cells, was unchanged in expression.
As expected from the immunochemical results, renin mRNA in the adrenals of the AS–/– male and female mice was very markedly increased, to 100 times wild-type mice. Expression of the Atr1b gene, which is the main Ang II receptor in the zG (27), was increased 7 times relative to wild type, whereas expression of the Atr1a gene, which is the Ang II receptor in the zF and zG (28), was only slightly increased.
Although, as indicated above, the heterozygous mice (AS+/–) have AS mRNA levels reduced to about half of wild-type, they have no morphological or physiological abnormalities and show no differences in the levels of expression of the genes that we tested in the AS–/– animals.
Discussion
We have generated and characterized AS-null mice (AS–/–) and have shown that they are unable to synthesize aldosterone. About 70% of the AS-null mice survive to weaning but have decreased body size, failure to thrive, hypotension, increased concentrations of plasma K+ and corticosterone, a decreased concentration of plasma Cl–, and very high plasma renin activity.
There are two well-known AS deficiencies in humans: corticosterone methyloxidase deficiency types I and II (29). Type I is caused by mutations in CYP11B2 that result in the expression of AS that has lost all of its activities. The type I patients have undetectable aldosterone levels, increased levels of 18-hydroxy-11-deoxycorticosterone, and reduced levels of 18-hydroxycorticosterone. The patients have severe salt loss, hypotension, hyperkalemia, hyponatremia, and very high plasma renin activity. They also have metabolic acidosis as neonates, fail to thrive in infancy, and show growth retardation in early childhood (30, 31). The type II form of AS deficiency in humans is characterized by low aldosterone and increased 18-hydroxycorticosterone levels. It is caused by mutations that impair the 18-hydroxylase and 18 methyl-oxidase activities of AS, but the 11?-hydroxylase activity is retained. These patients have a phenotype closely similar to the type I phenotype.
The AS-null mice share all the features of the type I human patients, including a very high plasma renin activity (45x wild-type level). Their plasma Ang II levels are increased to about 4x wild-type. Yet, the mice are hypotensive (BP about 14 mm Hg below wild type). Thus, the AS–/– mice, like type I corticosterone methyloxidase-deficient humans and MR-deficient mice (15), are unable to maintain a normal BP in the absence of aldosterone, despite their high plasma renin and increased Ang II levels. Nevertheless, it is clear that compensatory mechanisms that strongly activate the renin-Ang system (RAS) and up-regulate glucocorticoid production have been induced in the AS–/– mice. However, these compensations are not sufficient to normalize the plasma K+ and Cl– concentrations altered by the absence of aldosterone.
Comment is required on the survival of about 70% of our AS–/– pups and the absence of hyponatremia and salt wasting without salt supplementation compared with the absence of survival of either adrenalectomized animals or MR–/– mice (15) without supplementation. A possible explanation of this difference is that the AS–/– mice retain some control of sodium transport via up-regulated production of glucocorticoids that can act through the MR (32, 33, 34). This contrasts with the adrenalectomized animals, which are more handicapped because they cannot synthesize ligand, mineralocorticoid, or glucocorticoid, and the MR–/– mice, which lack the receptor.
Our data show that absence of aldosterone induces marked cellular changes in the adrenal cortex. The most easily detected response in the AS–/– mice is a massive increase in renin gene expression (100x wild-type level) in the adrenal cortex. This response is in accordance with previous work showing that in the adrenal cortex, the RAS plays a key role in controlling the secretion of aldosterone (10). Our data suggest that the easy detection of renin-containing cells in the adrenals of the AS–/– mice results from an increase in the amount of renin produced in individual renin producing cells possibly combined with an increase in their numbers. [Increases in the number of renin-containing cells are demonstrated in the kidneys of the aldosterone-deficient mice (Makhanova, M., G. Lee, M. L. Sequeira Lopez, R. A. Gomez, H.-S. Kim, N. Takahashi, and O. Smithies, unpublished data) and in mice lacking Ang (24), Ang-converting enzyme (35), or Atr1a (36).] Another indication of greatly increased RAS activity in the adrenals of the AS–/– mice is a 7-fold increase in the expression of Atr1b, the most abundant receptor of Ang II in the zG.
The absence of aldosterone in the AS–/– mice leads to several abnormalities in adrenal structure, including widening of the glomerulosa, an abnormal morphology of the fasciculata cells that have foamy and vacuolated cytoplasms caused by a greatly increased accumulation of lipid. This lipid accumulation is very similar to the adrenal lipid hyperplasia caused by absence of StAR, the StAR (37), or of Cyp11A1, the cholesterol side-chain cleavage enzyme (38). Interestingly, the mRNA levels of StAR and Cyp21A in the AS–/– mice are reduced to approximately 50% wild type, indicating down-regulation of the steroidogenic pathways.
Several features observed in the adrenals of the AS–/– mice indicate an increase in cell turnover. Thus, we found that the strongest EGFP signal, a marker for AS gene expression, was at the morphological boundary between the cortex and medulla, but these cells were highly apoptotic. Taken together, our data strongly support the hypothesis that increased RAS activity in the adrenal cortex induces the zG cells to undergo more cell turnover, presumably in an attempt to produce more aldosterone, and that these cells migrate through the cortex and enter apoptosis at the boundary between cortex and medulla.
In summary, we describe here a cellular and molecular characterization of aldosterone deficiency in mice and of the homeostatic changes induced in their adrenal glands. Despite a massive increase in plasma renin and Ang II, the mice are still hypotensive. The cortex of their adrenals shows a remarkable increase in the migration and apoptosis of cells that normally produce aldosterone. Renin-producing cells are easily demonstrated in the adrenal cortex in association with a 100-fold increase in adrenal renin mRNA. Therefore, the AS–/– mice should be valuable for studying how aldosterone regulates its own synthesis in the adrenals and affects the synthesis of renin in the adrenal glands and elsewhere.
Acknowledgments
We thank Kimberly Kluckman, Emily Riggs, and Gang Cui for technical assistance.
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