Sodium deficiency regulates rat adrenal zona glomerulosa gene expression

Abstract

Aldosterone is the primary adrenocortical hormone regulating sodium retention, and its production is under the control of the renin-angiotensin-aldosterone system (RAAS). In vitro, angiotensin II can induce aldosterone production in adrenocortical cells without causing cell proliferation. In vivo, a low-sodium diet activates the RAAS and aldosterone production, at least in part, through an expansion of the adrenal zona glomerulosa (zG) layer. Although these mechanisms have been investigated, RAAS effects on zG gene expression have not been fully elucidated. In this study, we took an unbiased approach to define the complete list of zG transcripts involved in RAAS activation. Adrenal glands were collected from 11-week old Sprague-Dawley rats fed either sodium-deficient (SDef), normal sodium (NS), or high-sodium (HS) diet for 72 hours, and laser-captured zG RNA was analyzed on microarrays containing 27 342 probe sets. When the SDef transcriptome was compared with NS transcriptome (SDef/NS comparison), only 79 and 10 probe sets were found to be up- and down-regulated more than two-fold in SDef, respectively. In SDef/HS comparison, 201 and 68 probe sets were up- and down-regulated in SDef, respectively. Upon gene ontology (GO) analysis of these gene sets, we identified three groups of functionally related GO terms: cell proliferation-associated (group 1), response to stimulus-associated (group 2), and cholesterol/steroid metabolism-associated (group 3) GO terms. Although genes in group 1 may play a critical role in zG layer expansion, those in groups 2 and 3 may have important functions in aldosterone production, and further investigations on these genes are warranted.

Figure 1.

FI and body weight changes. Eleven-week-old male SD rats were maintained on a 12-hour light, 12-hour dark cycle and given access to food/water ad libitum. After at least 1 week of acclimation by daily handling and weighing, the rats were randomly divided into 3 groups at day 0 and provided with diets containing different levels of sodium: SDef, NS, and HS. A, Amount of FI before and after diet change. FI was measured daily from the day before diet change (d −1) to the day of killing (d 3). Error bar, SEM. Statistical analysis was performed by two-way ANOVA with Holm-Sidak post hoc method. ***, P < .001. B, Body weight change. Body weights were measured daily throughout the experiment. Error bar, SEM; n.s., not significant.

Figure 2.

Serum aldosterone and CYP11B2 expression in the zG. After 13 or 72 hours of diet treatment of SDef, NS, and HS, rats were killed via rapid decapitation followed by blood and adrenal glands collection. A, Serum aldosterone among rat groups. Serum aldosterone levels were determined by RIA. Natural logarithm transformed serum aldosterone values were used in statistical analysis (two-way ANOVA with Holm-Sidak post hoc method). The boundary of the box indicates the 25th and 75th percentiles, and a line within the box marks the median. Whiskers below and above the box indicate the 10th and 90th percentiles, respectively. **, P < .01; ***, P < .001. B, qPCR for Cyp11b2. Laser-captured enriched populations of aldosterone-producing zG cells were used for Cyp11b2 qPCR. Fold changes were calculated by comparing with 72-hour-treated NS. Statistical analysis was performed by two-way ANOVA with Student-Newman-Keuls post hoc method using delta Ct value from qPCR. Error bar, SEM. **, P < .01; ***, P < .001. C, IHC, single-immunostaining for CYP11B2 with diaminobenzidine (brown) on 13-hour (top row)- and 72-hour (middle row)-treated adrenals as well as double-immunostaining for CYP11B2 with 5-bromo-4-chloro-3′-indolyphosphate (blue) and CYP11B1 with diaminobenzidine (brown) on 72-hour-treated adrenals (bottom row).

Figure 3.

Venn diagram showing the overlaps of differentially regulated genes in the three comparisons. Differentially regulated genes with more than two-fold difference and P < .05 were counted. Each circle of SDef group vs NS group (SDef/NS), SDef vs HS group (SDef/HS), and NS/HS shows the count of up-regulated genes and down-regulated genes in upper and lower side of circles, respectively.

Figure 4.

Heat map of genes with more than five-fold difference in expression level. The heat map was created for genes with greater than 5-fold changes with P < .05 in the comparison of SDef group vs HS group (SDef/HS) and SDef vs NS group (SDef/NS). Log2 signal intensities were shown in individual adrenal zG cells of 3 HS, 5 SDef, and 4 NS rats. Fold changes (FCs) in SDef/HS and SDef/NS comparisons were shown in the left and right side of the heatmap, respectively.

Figure 5.

Confirmation of microarray data using qPCR. Expression levels of 4 genes (A, Cdkn3; B, Rrm2; C, Nr4a1; and D, Scarb1) were determined using qPCR to confirm microarray data. Fold changes of HS and SDef were calculated by referring the value of NS. Error bar, SEM. *, P < .05; **, P < .01; ***, P < .001.