Abstract
In central osmoregulation, a 1–2% rise in plasma osmolality is detected by specialized osmoreceptors located in the circumventricular organs of the hypothalamus. A disturbance in this tightly regulated balance will result in either hyponatremia or hypernatremia, which are both common electrolyte disorders in hospitalized patients. Despite the high clinical importance of hypo- and hypernatremia and the fact that this vital process has been studied for many years, the genes and corresponding proteins involved in this process are just beginning to be identified. To identify novel genes involved in the (patho-)physiology of osmoregulation, we therefore employed haplotype association mapping on an aging group of 27 inbred mouse strains. Serum sodium concentrations were determined in all strains at 6, 12, and 18 mo of age, and high-resolution mapping was performed for males and females separately. We identified a total of five loci associated with the serum sodium concentration of which the locus on chromosome 14, containing only one known gene (Nalcn), showed the strongest correlation. Within this locus three different haplotypes could be distinguished, which associated with different average serum sodium levels. The association of Nalcn with sodium levels was confirmed by analysis of heterozygous Nalcn knockout mice, which displayed hypernatremia compared with wild-type littermates. Our study demonstrates that Nalcn associates with serum sodium concentrations in mice and indicates that Nalcn is an important novel player in osmoregulation.
Keywords: water homeostasis, sodium channels, central osmoregulation
in human osmoregulation, a 1–2% rise in plasma osmolality is detected by osmoreceptors located in the circumventricular organs of the hypothalamus. Within these organs, neuronal nuclei entitled the subfornical organ (SFO) and the organum vasculosum lamina terminalis (OVLT) mediate these responses, because they are able to detect changes in osmolality ex vivo (1, 2) and lesion of these nuclei in rats and dogs prevent generation of an adequate response with hypertonic stimuli (13, 22). With hypernatremia, activation of these nuclei result in two responses, 1) a thirst sensation and water intake, and 2) activation of a pathway leading to reduced urine production. For the latter, SFO/OVLT neurons project to the arginine vasopressin (AVP) producing magnocellular neurons of the supraoptic nucleus (SON), which in turn project to the posterior pituitary, where they release the antidiuretic hormone AVP into the bloodstream upon activation. Released AVP subsequently flows to the kidney where it binds the vasopressin-2 receptor and initiates a signaling cascade, eventually leading to an increase in aquaporin-2 protein expression in the apical membrane of collecting duct principal cells and increased water reabsorption from pro-urine (3, 4). With a normal functioning osmobalance, plasma osmolality is ∼290 mosmol/kgH2O, and sodium levels range between 136 and 144 mM in humans.
A disturbance in this tightly regulated water balance is manifested as a change in the serum sodium concentration annotated as hypernatremia or hyponatremia. Hypernatremia, defined as a serum sodium concentration >145 mmol/l, is a common electrolyte disorder that frequently develops in hospitalized patients and is associated with a higher mortality (8, 15, 19). Hyponatremia, defined as serum sodium concentration <136 mmol/l, has been recognized as the most common electrolyte abnormality encountered in clinical medicine. It is considered to be a comorbid condition requiring supportive treatment only. However, recent studies indicate that chronic hyponatremia is associated with attention deficits, gait disturbances, falls, and fractures (5, 18). Furthermore, it has been shown that hyponatremia is a risk factor for osteoporosis, myocardial infarction, and overall mortality independent of possible confounders. This was confirmed in a rat model for chronic hyponatremia where it was shown that hyponatremia for 3 mo significantly reduced bone mineral density by ∼30% compared with control rats (9, 24). The above-mentioned studies clearly show that hypernatremia and hyponatremia are important problems, not only in hospitalized patients but also in the general population.
Although of high clinical importance, most proteins involved in disturbed osmoregulation remain unidentified. In principle, a changed osmobalance can be due to changes in proteins directly regulating the osmobalance or can be a secondary effect due to changes in the regulation of the volume (sodium transport) balance.
Considering the importance of a proper osmoregulation and disturbances of it, we employed haplotype association mapping (HAM) on an aging group of mice to identify novel genes involved in the regulation of water homeostasis. HAM is a recently developed approach that utilizes high-density single nucleotide polymorphism (SNP) data from many inbred mouse strains to identify chromosomal haplotypes associated with phenotypic traits of interest (23). Here, we report the identification of several loci involved in the regulation of the osmobalance, including the locus only containing the Nalcn gene. Indeed, SNP variants in the Nalcn gene correlated with three levels of natremia and hypernatremia in heterozygous knockout mice for the Nalcn gene, confirming the involvement of NALCN as an important protein influencing the osmobalance.
MATERIALS AND METHODS
Animals and housing.
Twenty males and females from 27 different inbred strains (Table 1) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice used were housed in a climate-controlled facility with a 14-h:10-h light-dark cycle with free access to food and water throughout the experiment unless noted otherwise. After weaning, mice were maintained on a chow diet (Old Guilford 234A, Guilford, CT). Serum samples were taken at 6, 12, and 18 mo, and sodium concentrations were measured on a Beckman Synchron CX5 Chemistry Analyzer. All experiments were approved by The Jackson Laboratory's Animal Care and Use Committee.
Table 1.
The 27 inbred mouse strains used in the aging study
| 129S1/SvImJ | C57BL/10J | LP/J | PL/J |
| A/J | C57BLKS/J | MRL/J | PWD/PHJ |
| BALB/cByJ | C57L/J | NOD.B10Sn-H2b/J | RIIIS/J |
| BTBR T+ tf/J | CBA/J | NON/LtJ | SJL/J |
| BUB/BnJ | DBA/2J | NZO/H1LtJ | SM/J |
| C3H/HeJ | FVB/NJ | NZW/LacJ | WSB/EiJ |
| C57BL/6J | KK/J | P/J |
Haplotype association mapping.
Trait data at strain mean levels were input as vectors, and genotype data (SNPs) across multiple inbred mouse strains were input as a matrix. A hidden Markov model (HMM) was applied, fitting five states at each SNP, for the primary purpose of missing genotype imputation and for the secondary purpose of haplotype identification (21). At each SNP, the strain distribution pattern was determined using the HMM smoothed haplotype states (HMMpath). We computed regression-based test statistics to measure the strength of association between genotype and phenotype. Significance was estimated to detect groups with different mean phenotypes. The segregation of strains into genotypic groups varies widely over haplotype block; P values, not the test statistics, are compared between haplotype blocks. All P values were transformed using −log10(P value) in the scan plots. We managed the type I error rate for multiple testing due to genome-wide searching by using a family-wise error rate (FWER) control (26). We shuffled the strain label in the phenotype data and kept the genotype data intact. The minimum P value was recorded on each permutation, and percentiles of their distribution provided approximate multiple test-adjusted thresholds. The genome-wide type I error thresholds were estimated based on 1,000 permutation tests. Peaks corresponding to P value thresholds, adjusted for global significance, were defined as significant at an alpha of 0.05. Due to the close genetic relationship between genomes of inbred laboratory strains, HAM analysis has limited power to detect small genetic effects. Furthermore, FWER methods generally yield conservative results. To detect peaks that have small, but biologically relevant, genetic effects, we chose to relax the protection against type I errors and consider HAM peaks that exceeded an alpha of 0.63 to be suggestive evidence of genetic association. All analysis was done in the MATLAB computing environment (The Mathworks, http://www.mathworks.com), except for the imputation of missing genotypes.
Sequencing.
Genomic DNA was isolated from spleen. Primers were designed with the Primer3 program (primer3_www.cgi v 0.2) to cover the exons 25 through 44 of Nalcn and were ordered from Integrated DNA technologies (Coralville, IA). PCRs were performed on the genomic DNA. PCR products were sequenced in both directions on an Applied Biosystems 3730. Sequences were aligned using the sequence analysis program Sequencer 4.8 and compared with published sequences of the C57BL/6J obtained from Ensembl (Build 37).
Serum sodium concentration of heterozygous Nalcn knockout mice.
Heterozygous Nalcn knockout mice (12) were rederived at The Jackson Laboratory. With the mice at 12 wk of age, serum samples were collected and sodium concentrations were measured as described above.
Data analysis.
Differences between groups were tested by the Student's t-test corrected by the Bonferroni multiple-comparisons procedure. Differences were considered statistically significant for P < 0.05.
RESULTS
Haplotype association mapping.
The mean serum sodium levels for males and females of all strains are available at the Mouse Phenome Database (http://www.jax.org/phenome). The data, summarized in Fig. 1, show a large variability among the different strains and between sexes. Figure 2, A–F, shows the genome-wide scans for the serum sodium concentration at 6, 12, and 18 mo in males and females. With the imputed 63,222 SNP set with an average spacing of 40.53 Kb between each SNP, we obtained high-resolution mapping with small associated intervals. We did not identify any peaks above the stringent significance threshold of α = 0.05 but did observe several above the suggestive threshold (Table 2). The interval for each peak was examined with a second SNP set of 1.5 million SNPs, including all the SNPs from the first set, confirming the haplotype differences and boundaries. The intervals of most peaks were still large, containing many genes; the exception was the peak on chromosome 14 which had the strongest association.1 This locus contains only the distal part of Nalcn (exons 25–44), encoding a nonselective sodium leak channel. Within this locus, we can distinguish three different haplotypes (Table 3 and Fig. 3A) that associate with significantly different average serum sodium levels among the strains: strains with haplotype 1, 153.4 ± 3.2 mmol/l; strains with haplotype 2, 157.9 ± 4.8 mmol/l; strains with haplotype 3, 164.0 ± 3.9 mmol/l (Fig. 3B).
Fig. 1.
Serum sodium concentrations of all mouse strains at different ages. Mean (± SE) serum sodium concentrations (in mmol/l) for females and males of all mouse strains were measured at 12, 18, and 24 mo. A large variability in serum sodium concentration among the different strains used and between sexes can be observed.
Fig. 2.
Genome-wide haplotype association mapping in mice identifies 5 loci to be associated with changed serum sodium concentration. Genome-wide scans for female (A–C) and male (D–F) mice at 6 (A, D), 12 (B, E), and 18 mo (C, F). The 0.63, 0.10, and 0.05 alpha thresholds as determined by permutation testing are indicated. None of the loci reached significance, but 5 loci are above the suggestive threshold.
Table 2.
Summary of HAM peaks
| Age, mo | Sex | Chr | Locus, Mb* | Score | P Value | Genes in Interval |
|---|---|---|---|---|---|---|
| 6 | M | 5 | 114.1–114.4 | 3.64 | 2.27 × 10−4 | 8 genes |
| 7 | 107.2–109.0 | 3.76 | 1.75 × 10−4 | 34 genes | ||
| 12 | F | 14 | 55.9–56.3 | 4.42 | 3.77 × 10−5 | 24 genes |
| 18 | F | 12 | 114.6–114.8 | 3.83 | 1.47 × 10−4 | 22 genes |
| 14 | 123.5–123.7 | 4.53 | 2.98 × 10−5 | Nalcn |
HAM, haplotype association mapping; Chr, chromosome; M, male; F, female.
According to Ensembl build 37.
Table 3.
Strains arranged by the Chr14 locus haplotype
| Haplotype | Strains |
|---|---|
| 1 | BTBR, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, DBA/2J, LP/J, NON/LtJ, PL/J, PWD/PhJ, SJL/J |
| 2 | 129S1/SvImJ, A/J, BALB/cJ, BUB/BnJ, CBA/J, FVB/NJ, KK/HlJ, MRL/MpJ, NZW/LacJ, RIIIS/J, SM/J, SWR/J |
| 3 | C57L/J, NZO/HlLtJ, P/J |
Fig. 3.
Haplotype-specific single nucleotide polymorphism (SNP) pattern and serum sodium levels of the 3 variants in the Nalcn gene. A: map of the Chromosome 14 associated region including the genotyped SNPs and the 3 different haplotypes. The distal part of the Nalcn gene falls within the associated region. B: allele effect for each of the 3 haplotypes on the serum sodium levels in 18-mo-old females.
Characterization of Nalcn.
Sequencing of the coding regions present in each haplotype (exons 25–44) was performed on strains representing the three different haplotypes: C57BL/6J (haplotype 1), 129S1/SvImJ (haplotype 2), and C57L/J (haplotype 3). We identified seven synonymous SNPs (Cs), one nonsynonymous SNP (Cn), and three SNPs located in the splice sites (splice). Sequence differences are summarized in Table 4. The nonsynonymous SNP encodes a Serine (Ser) in haplotype 1 and a Threonine (Thr) in haplotypes 2 and 3. As there are three distinct haplotypes with significant differences in serum sodium levels in 18-mo-old females, one single SNP would not be able to explain this; at least two SNPs with an additive effect would be required: for example, an SNP identical in haplotypes 2 and 3 but different in haplotype 1, and in addition, an SNP unique to haplotype 3. Such a pattern exists in our sequence data: we found an amino acid difference unique to haplotype 1 (i.e., Ser instead of Thr), and three SNPs in splice sites unique to haplotype 3. Since the nonsynonymous SNP rs30499120 is the only common SNP between haplotypes 2 and 3, this SNP could cause the higher sodium levels in the strains with these haplotypes. For this reason we investigated the allele effect of this SNP on the serum sodium concentration in female and male mice at 6, 12, and 18 mo (Fig. 4). For mice with the T allele (Ser; haplotype 1), concentrations remain constant at 6, 12, and 18 mo; for mice with the A allele (Thr; haplotypes 2 and 3), concentrations increase over time. For females, this increase is already observed at 12 mo (P < 0.05) and increases even more by 18 mo (P < 0.0001). For males, the concentration is significantly different only at 18 mo (P < 0.0005).
Table 4.
Summary of Nalcn sequencing
| SNP ID | Location | Type | Haplotype 1 | Haplotype 2 | Haplotype 3 |
|---|---|---|---|---|---|
| intron 29 | splice | C | C | T | |
| intron 30 | splice | A | A | G | |
| rs46822409 | exon 38 | Cs | A | A | G |
| exon 38 | Cs | G | G | G | |
| intron 40 | splice | G | G | A | |
| exon 40 | Cs | G | G | A | |
| rs47158283 | exon 40 | Cs | A | G | A |
| rs462444506 | exon 41 | Cs | A | A | G |
| rs30322545 | exon 43 | Cs | G | A | G |
| exon 44 | Cs | C | C | T | |
| rs30499120 | exon 44 | Cn | T | A | A |
SNP, single nucleotide polymorphism.
Fig. 4.
SNP rs30499120-related effects on serum sodium levels in male and female mice at different ages. Of mice with the A or T SNP rs30499120 variant, the serum sodium concentrations are given for female (left) and male (right) mice at 6, 12, and 18 mo. It reveals that the A allele, coinciding with a Thr instead of a Ser at position 1699, leads to hypernatremia, which is more pronounced in female than in male mice.
Serum sodium concentration in heterozygous Nalcn knockout mice.
To confirm the association between Nalcn and sodium levels, we analyzed serum sodium concentration in wild-type and heterozygous Nalcn knockout mice. Heterozygous mice were used because homozygous Nalcn knockout mice die within 24 h after birth (12). At 12 wk of age, heterozygous mice displayed a significantly higher serum sodium concentration compared with wild-type mice (149.1 ± 0.8 vs. 146.5 ± 0.7; P < 0.02) (Fig. 5).
Fig. 5.
Mice heterozygous for Nalcn display an increased serum sodium concentration. At 12 wk of age, blood was taken from mice heterozygous for Nalcn and analyzed for their sodium levels. The heterozygous mice (het) showed a significantly higher serum sodium concentration compared with wild-type mice (wt) (149.1 ± 0.8 vs. 146.5 ± 0.7; P < 0.02).
DISCUSSION
In this study, our goal was to investigate the genetics of water homeostasis as measured by the serum sodium concentration in mice and to identify possible candidate genes that play an important role in this process. We used HAM, a novel phenotype-driven approach that uses high-density SNP data sets to search for associations between chromosomal regions and a particular trait of interest, to identify novel quantitative trait loci (QTL) associated with the serum sodium concentration in mice. This method has been successfully used not only to verify previously characterized QTL for high-density lipoprotein and gallstone phenotypes (16), but also to identify novel QTL or to refine the QTL and thereby narrow the number of candidate genes (11). Because HAM methodology is still in its infancy, the most appropriate multiple testing correction method to determine thresholds for significance is still unknown. We identified a total of five loci associated with the serum sodium concentration in mice by using a threshold of α < 0.63. We choose this threshold with the risk of type I errors as an explorative approach, realizing that any finding would need careful examination and confirmation using other methods, such as knockout models.
Genetic variations of Nalcn may explain the three different haplotypes.
Within each of the five serum sodium associated intervals, we identified as few as one or as many as 34 known genes. Among the intervals we associated with serum sodium, the chromosome 14 interval of 123.5–123.7 Mb, which contains only one known gene (Nalcn), showed the strongest correlation with the serum sodium concentration. We were able to distinguish three different haplotypes, and sequencing of the coding regions resulted in the identification of seven synonymous SNPs, one nonsynonymous SNP, and three SNPs located in splice sites. With three distinct haplotypes that associated with significant differences in serum sodium levels, we needed at least two SNPs with additive effects to explain the association.
The heterozygous Nalcn mice, expressing Nalcn from one allele only, were hypernatremic, indicating that a reduced Nalcn expression leads to hypernatremia. The identified SNPs could explain the significant differences in serum sodium levels between the three groups. The seven synonymous SNPs in Nalcn can lead to affected splicing or reduced mRNA stability, resulting in increased serum sodium concentration. The SNP associated with the Ser1699Thr modification is unique to haplotype 1 (rs30499120), and the three splice-site SNPs are unique to haplotype 3. Since the rs30499120 SNP is the only SNP that haplotypes 2 and 3 have in common, the substitution of Thr for Ser at position 1699 could underlie the increased serum sodium levels over time in mice with haplotype 2 or 3. This Thr1699Ser variant is located in the carboxy terminus of NALCN. Interestingly, Nalcn is related to voltage-gated sodium channels, and it has been shown that the carboxy terminus plays an important role in the inactivation kinetics of these channels, suggesting that this variation could affect the inactivation properties of Nalcn (10, 14).
SNP rs30499120, which is unique to haplotype 1, may explain the increase in serum sodium concentration associated with haplotypes 2 and 3. The unique SNPs in haplotype 3, may explain the additional increase in serum sodium concentration associated with haplotype 3. In fact, in haplotype 3, we found three unique SNPs that are located in splice sites of Nalcn. Thus, the additional increase in serum sodium concentration with haplotype 3, compared with haplotype 2, may be due to alternatively spliced Nalcn.
Potential physiological role of Nalcn in osmoregulation.
Nalcn, which encodes a member of the sodium/calcium channel family, was recently found to be responsible for a background sodium leak current in neurons, thereby controlling the membrane potential and setting the levels of neuronal excitability. It was shown that, unlike other members of the sodium/calcium channel family, NALCN forms a voltage-independent and nonselective cation channel. Furthermore, knockout mice lacking Nalcn displayed an impaired electrical discharge in spinal cord nerves (C4) along with severe respiratory problems which caused these mice to die within 24 h after birth (12). Although the study of Lu et al. (12) clearly illustrates the importance of Nalcn in neuronal function, the early neonatal fatality observed in Nalcn-deficient mice might mask other physiological roles of Nalcn. In our study, we observed increased serum sodium concentration in Nalcn heterozygous mice, indicating a novel and important role for Nalcn in maintaining serum sodium levels.
At present, it is unclear how variations in Nalcn lead to hypernatremia. Nalcn mRNA has been found in several regions of the brain, adrenal glands, heart, and pancreatic islands (20). However, whether functional NALCN is expressed in these tissues is unknown and remains to be established. Furthermore, additional investigation is needed to determine whether expression of Nalcn could influence heart rate, modulate neuronal AVP release and thirst sensation, or alter adrenal aldosterone release, any of which could possibly influence sodium balance and result in hypernatremia.
Potential involvement of non-Nalcn genes identified in osmoregulation.
Besides Nalcn on chromosome 14, we identified 34 other genes within each of the five serum sodium associated intervals. Although many genes have no reported associations with water homeostasis, some of them, such as Arap1 and P2y2r, have been linked to such activity. Arap1, located on chromosome 7, encodes a Type 1 angiotensin II receptor-associated protein, which has been implicated in recycling of the angiotensin II receptor (AT1); mice with a proximal tubule-specific overexpression of Arap1 develop hypertension and kidney hypertrophy (6, 7). P2y2r, located on chromosome 7, encodes the P2Y subtype of purinergic receptors and has been linked to hypertension in Japanese men (25). Furthermore, it has been shown that mice lacking the P2y2r gene display an increased urinary concentration due to a higher AQP2 protein expression, and display a salt-resistant arterial hypertension (17, 28). More recently, it was shown that dDAVP treatment of mouse collecting duct cells caused P2Y2R translocation to the apical and basolateral membrane and that activation of exogenous P2Y2R in Xenopus oocytes decreased membrane AQP2 expression and AQP2-mediated water permeability (27). Taken together, these studies indicate that, of the genes identified with our HAM analysis as likely to be involved in osmoregulation, Arap1 and P2y2r are the most likely chromosome 7 genes to affect water homeostasis. These results underscore the value of our HAM analyses to identify genes involved in water homeostasis. It remains to be established which of the genes, identified in intervals on chromosomes 5 and 12 and in the 55.9–56.3 Mb interval on chromosome 14, may be involved in regulating the water balance.
GRANTS
This work was funded by National Institutes of Health Grants DK-069381 (R. Korstanje) and AG-25707. P. M. T. Deen is a recipient of VICI Grant 865.07.002 of the Netherlands Organization for Scientific research (NWO). This study was further supported by grants from NWO (865.07.002), the Coordination Theme 1 (Health) of the European Community's 7th Framework Program RTN aquaglyceroporins (number 035995-2), the UMC St Radboud (2006-35) to P. M. T. Deen and by the Disease Models & Mechanisms travelling fellowship to A. P. Sinke.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
Supplementary Material
ACKNOWLEDGMENTS
We thank Dana Godfrey and Sue Grindle for excellent technical assistance, Joanne Currer for writing assistance, and Jesse Hammer for preparation of the figures.
Footnotes
The online version of this article contains supplemental material.
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