Aldosterone Postnatally, but not at Birth, Is Required for Optimal Induction of Renal Mineralocorticoid Receptor Expression and Sodium Reabsorption

Aldosterone is dispensable at birth but rather crucial in the postnatal period for optimal induction of the mineralocorticoid signaling pathway and for sodium homeostasis.

Abstract

Sodium wasting during the neonatal period is the consequence of a physiological aldosterone resistance, related to a low renal mineralocorticoid receptor (MR) expression at birth, both in humans and mice. To investigate whether aldosterone is involved in the neonatal regulation of MR expression, we compared aldosterone and corticosterone levels and renal MR expression by quantitative real-time PCR, between aldosterone synthase (AS) knockout, heterozygous, and wild type (WT) mice, at birth and postnatal d 8. Analysis of MR transcripts showed a similar expression profile in all genotypes, demonstrating that the lack of aldosterone does not modify either the low renal MR expression at birth or its postnatal induction. However, mRNA levels of the α-subunit of the epithelial sodium channel, a MR target gene, were significantly higher in WT compared with AS knockout mice, both at birth and postnatal d 8, despite high corticosterone levels in AS knockout mice, indicating that aldosterone is required for optimal renal induction of the epithelial sodium channel. Using organotypic cultures of newborn WT kidneys, we confirmed that aldosterone does not regulate MR expression at birth, but is instead capable of increasing MR expression in mature kidneys, unlike dexamethasone. In sum, we demonstrate both in vivo and in vitro, that, whereas aldosterone has no significant impact on renal MR expression at birth, it is crucial for optimal MR regulation in postnatal kidneys and for appropriate hydroelectrolytic balance. Understanding of MR-regulatory mechanisms could therefore lead to new therapeutic strategies for the management of sodium loss in preterms and neonates.

In the neonatal period, the human kidney displays a tubular immaturity, with associated sodium wasting, negative sodium balance, and impaired water reabsorption (1). This inability to maintain homeostatic function is accentuated in preterm infants (2–4) and represents a critical problem that pediatricians have to deal with. Therefore, a better understanding of water and sodium regulation during this specific developmental period is of major importance to propose new therapeutic strategies for the management of preterm infants.

Renal sodium reabsorption is mainly controlled by aldosterone, a steroid hormone synthesized in the adrenal gland zona glomerulosa, secondary to renin stimulation via angiotensin II and to potassium stimulation (5). In the distal nephron, aldosterone, by binding to its receptor, the mineralocorticoid receptor (MR), a transcription factor (6), tightly regulates the expression and activity of several transporting proteins implicated in sodium homeostasis, including the α-subunit of the epithelial sodium channel (αENaC) (7). The selectivity of the mineralocorticoid-signaling pathway in the epithelial cells is controlled at a prereceptor level by the 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2), which metabolizes cortisol (or corticosterone in rodents) into inactive compounds, incapable of MR binding (8, 9). We have previously demonstrated that the neonatal sodium wasting is related to a physiological transient renal aldosterone resistance (10). We have also established that this physiological aldosterone resistance is associated in both mice and humans with a low renal MR expression at birth, both at the mRNA and protein level (11). This low renal MR expression in newborns is followed by a significant increase in the postnatal period, with a complete renal tubular expression developed at 8 postnatal days in mice and during the first year of life in humans, paralleling renal maturation. The underlying mechanisms, responsible for the low renal MR expression at birth and its postnatal increase, are currently unknown. Because aldosterone levels are very high at birth and have a tendency to decrease in the postnatal period, mirroring MR expression (11), we hypothesized that these high hormonal levels could contribute to the low neonatal renal MR expression. To investigate the role of aldosterone on neonatal MR regulation, we used the aldosterone synthase (AS) knockout mouse model (AS⁻/⁻). These transgenic mice were originally generated by Kim and associates (12), using standard gene-targeting methods, with a final construct lacking exons 1, 2, part of exon 3, and introns 1 and 2 of the AS gene, and have undetectable levels of plasma aldosterone. AS⁻/⁻ genotype is compatible with fetal development, but newborns fail to thrive postnatally and about 30% die between d 7 and d 28 (12). Adult AS⁻/⁻ mice are small, weigh 25% less than wild-type (WT) animals, and have low blood pressure, abnormal electrolyte homeostasis (increased plasma concentrations of K⁺, Ca²⁺, and Mg²⁺, decreased concentrations of HCO3⁻ and Cl⁻ but no difference of plasma Na⁺ level under normal diet) and altered water metabolism (higher urine output, decreased urine osmolality, and impaired urine concentrating and diluting ability). Higher levels of plasma corticosterone and strong activation of the renin-angiotensin system are observed. In contrast, AS⁺/⁻ mice have normal plasma aldosterone concentrations, normal blood pressure, and no electrolyte disturbances (13). To evaluate whether renal MR expression in the neonatal period is dependent on aldosterone, we have quantified and compared MR mRNA steady-state levels in the kidneys of AS⁻/⁻, AS⁺/⁻, and WT littermates, using quantitative real-time PCR. To determine whether maternally derived aldosterone exposure in utero modifies MR expression at birth, we conducted two breeding strategies, using pups derived from either AS^−/− or AS^+/− mice. We have also analyzed the expression of other actors of the mineralocorticoid [11βHSD2, epithelial sodium channel (αENaC), and glucocorticoid receptor (GR)]-signaling pathways, and measured hormonal status. Our results were subsequently confirmed in vitro, using organotypic cultures of WT newborn murine kidneys. We demonstrate that, whereas aldosterone is dispensable for MR regulation at birth, it is pivotal for optimal induction of renal MR expression and sodium homeostasis in the postnatal mature kidney, effects that cannot be compensated by the glucocorticoid-signaling pathway.

Materials and Methods

Animals and breeding strategy

All experiments were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee. AS⁻/⁻ mice, previously described (12), were generated on a 129 background and backcrossed over 15 generations or more onto the C57Bl6/J strain (The Jackson Laboratory, Bar Harbor, ME). Mice were genotyped by real-time PCR (7900HT; Applied Biosystems, Foster City, CA) using Taqman probes for a sequence in the Cyp11b2 gene and for a portion of the gene contained in the neomycin resistance cassette, as previously described (14). Animals were housed in a temperature-controlled facility with a 12-h light, 12-h dark cycle. AS⁻/⁻ female mice were mated with AS⁻/⁻ male mice to obtain AS⁻/⁻ littermates. AS⁺/⁻ female mice were mated with AS⁺/⁻ male mice to obtain AS⁻/⁻, AS⁺/⁻, and WT littermates. Mice were killed at birth (D0) and at postnatal d 8 (D8) by decapitation. Kidneys were immediately collected, snap-frozen in liquid nitrogen, and stored at −80 C until analysis.

Hormonal analyses

Trunk blood was collected into dipotassium-EDTA tubes (Microvette CB K2E; Sarstedt AG & Co., Numbrecht, Germany) and centrifuged at 6000 rpm for 5 min, after which plasma was stored at −80 C until assay. Aldosterone was determined as previously described (14) using a RIA using [¹²⁵I]aldosterone (MP Biomedicals, Irvine CA), a primary antibody to aldosterone (National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone & Peptide Program, Torrance CA), and a secondary antirabbit γ-globulin antibody (Linco Research, St. Charles, MO). Corticosterone was measured using a commercially available RIA kit (ImmuChem Double Antibody Corticosterone Kit, MP Biomedicals) as described in Ref. 14.

Organotypic cultures

Kidneys were collected from WT mice on D0 or at D8. Each D0 kidney was incubated for 15 min in 150 μl of accutase (PAA Laboratories, Les Mureaux, France), and D8 kidneys were incubated for 1 h in 300 μl of trypsin (Invitrogen, Cergy-Pontoise, France). Then, kidneys were manually dissected with a needle. Homogenates were pooled and centrifuged for 3 min at 300 × g, supernatant was withdrawn, and cellular pellet was resuspended in a specific epithelial medium described below. Cellular suspensions were then seeded on collagen I-coated 12-well plates (Collagen I from Institut Jacques Boy, France), and routinely cultured for 7 d at 37 C in a humidified incubator gassed with 5% CO₂ within an epithelial medium composed of DMEM/Ham's F12 (1:1), 2 mm glutamine, 50 nm dexamethasone, 50 nm sodium selenite, 5 μg/ml transferrin, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 2 nm T3, 100 U/ml penicillin/streptomycin, 20 mm HEPES, pH 7.4, 5% dextran charcoal-treated serum, and 1% amphotericin B (the latter added to the medium for the first 48 h of culture only).

To investigate aldosterone and corticosteroid action, the epithelial medium was replaced at d 7 of culture by a minimal medium (MM), which has the same composition as the epithelial medium but lacks dexamethasone and dextran charcoal-treated serum. After 24 h in MM, either ethanol or aldosterone or dexamethasone, as well as spironolactone, was added to the medium for 24 h as indicated.

RT-PCR and quantitative real-time PCR

Total RNA was extracted from tissues or cells with the TRIZOL reagent (Invitrogen) according to the manufacturer's recommendations, and RNA was thereafter processed for RT-PCR, as previously described (11). Total RNA (1 μg) isolated from frozen samples, was subjected to deoxyribonuclease I Amplification Grade treatment (Invitrogen) and then reverse transcribed by use of High-Capacity cDNA reverse transcription kit from Applied Biosystems (Life Technologies, Villebon sur Yvette, France). Samples were diluted 10-fold after which 1/20 of the reverse transcription reaction was used for quantitative RT-PCR (qRT-PCR) using the Fast SYBR Green Master Mix (Applied Biosystems) containing 300 nm of specific primers (Supplemental Table 1 published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). qRT-PCR was carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems). Reaction parameters were as follows: 95 C for 20 sec, then 40 cycles at 95 C for 1 sec and 60 C for 20 s. For preparation of standards, amplicons were purified from agarose gel and subcloned into pGEMT-easy plasmid (Promega, Charbonnières-les-Bains, France) and sequenced to confirm the identity of each sequence. Standard curves were generated using serial dilutions of linearized standard plasmids, spanning 6 orders of magnitude. Standard and sample values were amplified in duplicate and analyzed from three independent experiments. Ribosomal 18S was used as an internal control for data normalization. Relative expression of a given gene is expressed as the ratio attomoles of specific gene/femtomoles of 18S. Results are mean ± sem and represent the relative expression compared with that obtained in AS⁻/⁻ mice kidneys at D0, which was arbitrarily set at 1. Supplemental Table 1 indicates primer sequences of genes analyzed by qRT-PCR.

Immunocytochemistry

Cells were fixed in 10% buffered formol in PBS (pH 7.3) for 15 min and then washed three times in PBS before processing for immunocytochemistry, as previously described (11). For immunocytochemical analyses, we used the monoclonal anti-MR antibody clone 6G1, generously provided by Dr. Gomez-Sanchez (University of Mississippi,) (15), at the dilution of 1:40.

Statistical analyses

Results are expressed as mean ± sem of at least three independent analyses with at least six samples for each developmental stage. Statistical analyses were performed using a nonparametric Mann Whitney test (Prism4, Graphpad Software, Inc., San Diego, CA), with significant threshold at 0.05.

Results

Breeding strategy

We conducted a specific breeding strategy to obtain four different newborn mice with various genotypes and distinct developmental endocrine patterns. We obtained AS⁻/⁻ pups that had never been exposed to aldosterone, by mating female AS⁻/⁻ mice with AS⁻/⁻ male mice. We obtained AS⁻/⁻ pups that had been exposed to maternal aldosterone, through transplacental crossing (16), by mating AS⁺/⁻ female mice with AS⁺/⁻ male mice, which also generated AS⁺/⁻ littermates and WT newborn mice. No difference was observed between genotypes regarding weight at birth (1.23 ± 0.07, 1.39 ± 0.02, 1.38 ± 0.04, 1.42 ± 0.11 g, respectively), and at D8 (3.51 ± 0.23, 3.54 ± 0.19, 4.11 ± 0.17, 4.02 ± 0.27 g, respectively). Kidneys were collected at D0 and at D8. No renal structural abnormality was observed.

Aldosterone and corticosterone levels

Aldosterone and corticosterone levels were measured in AS⁻/⁻ and WT newborn mice at the time of kidney retrieval (Fig 1). As expected, aldosterone was undetectable in AS⁻/⁻ mice both at D0 and D8. In WT mice, mean aldosterone level at birth was 466 ± 245 pg/ml and decreased at D8 (94 ± 38 pg/ml), although the difference did not reach significance (P = 0.18). Interestingly, we found that, in AS⁻/⁻ animals both at birth and at D8, very high levels of corticosterone (374 ± 317 and 681 ± 376 ng/ml, respectively) were detected in comparison with WT mice of the same developmental stage, particularly at D8 (P = 0.04). Analogous high levels of corticosterone have also been reported in AS⁻/⁻ adult mice on normal sodium diet (13). One could hypothesize that these high corticosterone levels are secreted to compensate for aldosterone deficiency.

Fig. 1.

Aldosterone and corticosterone levels: Aldosterone and corticosterone levels were measured in AS⁻/⁻ [knockout (KO)] and WT mice at D0 and at D8. Results are expressed as the mean ± sem of six different samples. *, P < 0.05.

MR expression

MR transcript levels were measured by qRT-PCR in the kidneys of all four groups of animals at D0 and D8 (Fig 2). No difference was evident at birth between genotypes, therefore suggesting that the low renal MR expression during the neonatal period is independent of aldosterone exposure. In all conditions, a significant increase in renal MR mRNA expression level was observed in all animals at D8, demonstrating that aldosterone does not intervene into the postnatal up-regulation of renal MR expression. However, we found a significant difference (P < 0.05) in D8 state levels of MR transcripts between AS⁻/⁻ and WT mice, suggesting that aldosterone may regulate MR expression only in postnatal developed kidneys, when complete tubular maturation is achieved.

Fig. 2.

MR expression: MR mRNA expression was measured in the kidneys of AS⁻/⁻ [knockout (KO)], AS⁺/⁻ heterozygous (HET)], and WT mice at D0 and at D8. Relative mRNA levels were determined by qRT-PCR in at least four different samples of each genotype and developmental stage. Results, expressed as the ratio of attomoles of specific gene per fentomoles of 18S, are means ± sem of three independent experiments and correspond to the relative expression compared with that obtained in AS⁻/⁻ mice kidneys at D0, which was arbitrarily set at 1. *, P < 0.05; ***, P < 0.001.

11βHSD2 and αENaC expression

We have previously demonstrated in WT mice (11), a parallel evolution throughout renal development between MR, and both the 11βHSD2 enzyme, which confers mineralocorticoid selectivity, and αENaC, a prototypal MR target gene. Because corticosterone levels were very high in AS⁻/⁻ mice, we wondered whether the expression profiles of 11βHSD2 and αENaC would be modified in the four different genotypes. It has indeed been suggested that the glucocorticoid pathway could be implicated in the regulation of these two genes (17–19). The question raised was whether corticosterone could compensate for aldosterone deficiency in AS⁻/⁻ mice by inducing αENaC expression via binding to the GR. The latter is a transcription factor closely related to MR and also known to activate gene expression implicated in sodium transport (18, 19). 11βHSD2 and αENaC mRNA expression were therefore analyzed at D0 and D8 in all genotypes. 11βHSD2 displayed an expression profile identical to that of the MR, with a low expression at birth and a significant increase in the postnatal period, independent of aldosterone status (Fig 3A). On the contrary, αENaC mRNA expression profile appeared strikingly different (Fig 3B). Indeed, αENaC mRNA levels were very low in AS⁻/⁻ mice both at D0 and D8, with no significant postnatal increase. αENaC expression evolved in parallel with aldosterone levels, with a 1.5-fold and a 2-fold increase at birth in AS⁺/⁻ and WT mice, respectively, and a 2.5-fold and 3-fold induction at D8 in these same genotypes, compared with levels in AS⁻/⁻ mice at D0. Of interest, at D0, levels of αENaC transcripts in AS⁻/⁻ newborns, originating from AS⁺/⁻ × AS⁺/⁻ mice, and thus prenatally exposed to aldosterone, were significantly higher than in nonexposed AS⁻/⁻ x AS⁻/⁻ littermates, whereas αENaC mRNA levels were identical at D8 in these animals. These results strongly suggest that maternally biosynthesized aldosterone may have a significant impact on fetal kidney αENaC expression. Indeed, at D8, when circulating aldosterone is no longer detectable in AS⁻/⁻ mice born from AS⁺/⁻ mothers, αENaC mRNA levels do not exhibit postnatal induction as happens in AS⁺/⁻ and WT animals. Therefore, it appears that aldosterone is instrumental for optimal renal induction of αENaC and subsequently for sodium tubular reabsorption in the neonatal period.

Fig. 3.

11βHSD2 and αENaC expression: 11βHSD2 (A) and αENaC (B) mRNA expression were measured in the kidney of AS⁻/⁻ knockout (KO), AS⁺/⁻ heterozygous (HET), and WT mice on D0 and at D8. Relative mRNA levels were determined by qRT-PCR in at least four different samples of each genotype and developmental stage. Results, expressed as the ratio of attomoles of specific gene per fentomole of 18S, are means ± sem of three independent experiments and correspond to the relative expression compared with that obtained in AS⁻/⁻ mice kidneys at D0, which was arbitrarily set at 1. NS, Not significant P = 0.66; ***, P < 0.001.

Furthermore, it becomes evident that despite the high levels of corticosterone observed in AS⁻/⁻ mice and their substantial levels of GR expression (see Supplemental Fig. 1), glucocorticoid signaling cannot functionally compensate the lack of mineralocorticoid signaling.

Organotypic cultures

We next decided to establish a reliable cell-based model to further investigate the hormonal regulatory mechanisms of renal MR expression in the neonatal period. We thus chose to grow organotypic cultures of WT newborn (OCD0) and postnatal-d 8 (OCD8) mouse kidneys. As presented in Fig 4A, after manual kidney dissection, the nephronic tubules and glomeruli can be clearly visualized in the epithelial medium. Thereafter, epithelial-like tubular cells start to proliferate from nephronic fragments attached to the collagen I-coated plates. After 7 d of culture, a full layer of renal tubular cells is observed with the typical features of epithelial cells, most notably the capacity to form domes. Moreover, as shown in Fig 4B, we were unable to immunodetect MR protein in OCD0 after 7 d of culture, whereas a specific immunolabeling was readily detected in the epithelial-like tubular cells in OCD8, just like that observed in WT kidneys (11). The cell immunolabeling was intracytoplasmic in the absence of aldosterone (control), or in the presence of a MR antagonist, spironolactone (A-7 + Spiro-6), whereas aldosterone alone (A-7) induced a clear nuclear translocation of the activated MR. These results suggest that primary cultures of renal cells maintain the capacity to express MR protein and therefore constitute a valuable and reliable experimental cell-based model to investigate the impact of aldosterone (or corticosteroid hormone) exposure on neonatal (D0 and D8) control of renal MR expression.

Fig. 4.

OCD0 or OCD8 A, Organotypic cultures were grown in epithelial medium for 7 d. After dissection, single glomeruli and nephrons can be individualized in the medium. On the first day of culture (1) epithelial-like tubular cells start growing. After 7 d of culture (7) a layer of epithelial-like tubular cells is observed. These cells demonstrate specific features of epithelial cells and have the capacity to form domes (arrow). B, Immunodetection of MR protein in organotypic cultures, after 7 d of culture in epithelial medium for OCD0, or after 7 d of culture in epithelial medium followed by 24 h in MM and 24 h in either MM (Control), MM + Aldosterone 10⁻⁷ m (A-7) or MM + Aldosterone 10⁻⁷ m and Spironolactone 10⁻⁶ m (A-7+Spiro-6) treatment for OCD8. Immunoreactive MR is absent in OCD0, but still detectable in epithelial-like tubular cells of OCD8 after 1 wk of culture. MR immunostaining is cytoplasmic in the absence of aldosterone (Control) or in the presence of the MR antagonist, spironolactone, (A-7+Spiro-6). MR expression in the epithelial cells is nuclear in the presence of aldosterone (A-7). C, Relative MR mRNA expression was determined in OCD0 and OCD8 after 7 d of culture in epithelial medium, followed by 24 h in MM and 24 h in either MM (Control), MM + Aldosterone 10⁻⁷ m (A-7), or MM + dexamethasone 10⁻⁷ m (D-7) treatment. Results, expressed as the ratio of attomoles of specific gene per fentomole of 18S, are means ± sem. The control condition was chosen as our statistical reference.

Impact of aldosterone on gene expression in organotypic cultures

To explore the impact of mineralocorticoid and glucocorticoid hormones on neonatal renal mRNA expression, we incubated OCD0 and OCD8 kidneys with various steroids (aldosterone or dexamethasone). No variation in MR mRNA levels was observed in OCD0 after 24 h treatment with either hormone (Fig. 4C), providing additional support for the lack of corticosteroid hormone effects on MR expression when comparing D0 AS⁻/⁻ and WT mice (see Fig. 2). However, our cell model was sensitive to glucocorticoid but not aldosterone action because dexamethasone, a steroid compound that is not submitted to metabolic conversion by the 11βHSD2 enzyme (20), was able to induce αENaC expression (data not shown), most likely through GR activation. Indeed, the absence of MR protein in OCD0 explains why aldosterone is inefficient in inducing αENaC expression. Finally, in sharp contrast, in OCD8 cultures, clearly expressing MR protein, aldosterone, but not dexamethasone, was able to induce a significant 2-fold increase of MR transcripts (P < 0.01) (Fig 4C). This result corroborates the in vivo findings obtained in mouse models, revealing a significantly higher MR mRNA expression in WT than in AS⁻/⁻ mice, particularly at D8.

Discussion

In the present paper, we demonstrate both in vivo and in vitro, that, at birth, aldosterone has no impact on the renal expression of its specific MR. Physiologically, renal MR expression is extremely low during the neonatal period, both in mice and humans, and increases drastically during the postnatal period, in parallel with other actors of the mineralocorticoid pathway such as 11βHSD2, which confers mineralocorticoid selectivity, and αENaC, a prototypal MR target gene required for apical sodium entry and transepithelial reabsorption (11). Herein, we show that the lack of aldosterone biosynthesis observed in AS⁻/⁻ mice does not modify MR expression profile at this specific period of development, characterized by a persistent low expression at birth and a postnatal induction at D8. Similarly, aldosterone withdrawal or administration does not induce MR mRNA and protein expression in organotypic cultures of newborn kidneys collected on D0.

Interestingly, unlike the MR and 11βHSD2 mRNA expression profiles, which were independent of aldosterone status, αENaC mRNA levels positively correlated with the degree of aldosterone exposure, following the sequence AS⁻/⁻ less than AS⁺/⁻ < WT. Indeed, in the absence of aldosterone (in AS⁻/⁻ newborns from AS⁻/⁻ mothers, and in AS⁻/⁻ 8-d-old mice), αENaC transcripts levels were low at birth and did not increase postnatally.

The low, yet detectable, αENaC mRNA levels measured at birth in AS^−/− mice, in the absence of aldosterone and concomitant with a very low MR expression, emphasize the redundancy of gene expression regulation. Indeed, the mineralocorticoid pathway is not the sole regulator of αENaC expression. It can also be controlled by other transcription factors, such as the GR (21). We have previously demonstrated that GR is expressed in the neonatal kidney both at the mRNA and protein levels and is detected in the nuclei of the cortical collecting duct cells, consistent with a functional GR-mediated signaling pathway (11). GR is also detected in the kidney of AS⁻/⁻ newborns. Therefore the glucocorticoid signaling pathway could account for these detectable αENaC mRNA levels at birth.

However, in comparison, the abundance of αENaC mRNA presented a strict positive relationship with aldosterone status, with a significant increase in αENaC mRNA levels in AS heterozygous and WT newborns compared with AS⁻/⁻ mice at D0, associated with a further increase at D8, as previously demonstrated (11). Moreover, AS⁻/⁻ mice born from AS heterozygous mothers and thus exposed to aldosterone during fetal life, presented with higher αENaC mRNA levels at birth compared with AS⁻/⁻ newborns from AS⁻/⁻ mothers. In addition, ENaC transcript levels in the kidneys of the former animals decreased during the postnatal period along with the clearance of maternal aldosterone. These results confirm the major role of aldosterone in sodium homeostasis during the neonatal period, which cannot be compensated by the activation of glucocorticoid signaling pathway, despite the high corticosterone levels measured in AS⁻/⁻ newborn mice and the substantial GR expression. This lack of compensation could also be the consequence of variability in GR potency at this specific period of development (22). These findings are in accordance with Lee et al. (12) who have reported the death of one third of AS⁻/⁻ mice between 7 d and 28 d (corresponding to the period of complete kidney maturation and full renal MR expression), from dehydration and failure to thrive. The surviving mice presented with hypotension, hyperkalemia, and difficulty to concentrate urine, exacerbated under a sodium-restricted diet (23), even in the presence of high corticosterone levels and subnormal renal GR expression (12, 13). This is reminiscent of MR knockout newborns that present similar symptoms and die of dehydration at 8 postnatal days (24). Both transgenic mouse models can be rescued by sodium supplementation in the neonatal period (13, 25). These observations are also reminiscent of human infants carrying heterozygous inactivating MR mutations (26) or with AS deficiency (27–29), who present with failure to thrive and inability to reabsorb sodium in the postnatal period, thus requiring sodium supplementation to survive. Therefore, both aldosterone and MR appear critical for sodium reabsorption and maintenance of sodium and water homeostasis in the postnatal period and cannot be compensated by a functional glucocorticoid signaling pathway during this specific period of renal development.

It seems particularly interesting that all the components of the renal mineralocorticoid signaling pathway (MR, 11βHSD2 and αENaC) are physiologically down-regulated during the first days of life, both in mouse and human newborns. Therefore, these mechanisms concur to impair sodium reabsorption and facilitate sodium wasting, this physiological condition being of particular importance during these first days, regardless of aldosterone status. This dissociation between aldosterone and mineralocorticoid signaling pathway activation could constitute a renal protection mechanism preventing inappropriate sodium retention. Alternatively, high physiological aldosterone concentrations could potentially be required for rapid nongenomic effects, as previously suggested (30–32). Nevertheless, these observations could explain why deficiency of either aldosterone biosynthesis or MR expression becomes critical after the first week of life only, both in mice and humans.

Interestingly, at variance with D0, we demonstrated that renal MR expression becomes regulated by aldosterone at D8, most likely once MR mRNA and protein amounts reach threshold levels in mature kidneys. Indeed, we observed a significant increase of MR mRNA levels at D8 between AS⁻/⁻ and WT mice. These results were corroborated by organotypic culture experiments conducted on D8 WT kidneys, where MR mRNA levels were stimulated by aldosterone but not by dexamethasone. Unlike D0, MR protein expression is readily detected in WT kidneys at D8 and is maintained after 1 wk of organotypic culture. Therefore these results could be consistent with a positive autoregulatory process. Other authors have already suggested an aldosterone-dependent MR autoregulation (33, 34). The implication of MR in mediating these aldosterone-regulatory effects was established by using the MR antagonist spironolactone, (33), or by knocking down MR by small interfering RNA (34). Whether this effect is mediated by direct binding of activated MR on the regulatory region of the MR gene or involves intermediate transcription factors, such as Sp-1 or AP-2, as suggested by Zennaro et al. (33), remains to be further investigated. We therefore propose that aldosterone contributes to MR regulation through a positive homologous regulatory loop, most likely via MR. This autoregulation of renal MR expression is not functional at birth owing to the lack or very low abundance of MR protein in the newborn kidneys.

Finally, our results emphasize the multiple mechanisms (in both the mineralocorticoid and glucocorticoid signaling pathways), conserved among species, which synergistically contribute to maintain physiological sodium wasting and weight loss in every newborn.

Further investigations of MR-regulatory mechanisms, particularly in the neonatal period, could improve our understanding of the physiological mineralocorticoid resistance observed at birth and might open pharmacological options for the management of sodium wasting in preterms, the most concerned by this pathology.