Targeted disruption of the Kcnj5 gene in the female mouse lowers aldosterone levels

Abstract

Aldosterone is released from adrenal zona glomerulosa (ZG) cells and plays an important role in Na and K homoeostasis. Mutations in the human inwardly rectifying K channel CNJ type (KCNJ) 5 (KCNJ5) gene encoding the G-coupled inwardly rectifying K channel 4 (GIRK4) cause abnormal aldosterone secretion and hypertension. To better understand the role of wild-type (WT) GIRK4 in regulating aldosterone release, we have looked at aldosterone secretion in a Kcnj5 knockout (KO) mouse. We found that female but not male KO mice have reduced aldosterone levels compared with WT female controls, but higher levels of aldosterone after angiotensin II (Ang-II) stimulation. These differences could not be explained by sex differences in aldosterone synthase (Cyp11B2) gene expression in the mouse adrenal. Using RNAseq analysis to compare WT and KO adrenals, we showed that females also have a much larger set of differentially expressed adrenal genes than males (395 compared with 7). Ingenuity Pathway Analysis (IPA) of this gene set suggested that peroxisome proliferator activated receptor (PPAR) nuclear receptors regulated aldosterone production and altered signalling in the female KO mouse, which could explain the reduced aldosterone secretion. We tested this hypothesis in H295R adrenal cells and showed that the selective PPARα agonist fenofibrate can stimulate aldosterone production and induce Cyp11b2. Dosing mice in vivo produced similar results. Together our data show that Kcnj5 is important for baseline aldosterone secretion, but its importance is sex-limited at least in the mouse. It also highlights a novel regulatory pathway for aldosterone secretion through PPARα that may have translational potential in human hyperaldosteronism.

Keywords: GIRK4 K channel; KCNJ5 gene; RNAseq; aldosterone; knockout mice.

Figure 1. Representative sections of male and female WT (^+/+) and KO (^−/−) mouse adrenals stained for DAB2

Figure 2. YP11B2 and KCNJ5 gene expression in laser captured adrenal zones

(A) DAB2-stained adrenal section before (A) and after (B) laser capture of specific adrenal zones. Relative gene expression of Cyp11B2 (C) and KCNJ5 (D). Bars are means of two experiments in laser captured tissue. (E) (Inset) shows gel separation of qPCR products for (D). Abbreviations: ND, not detected by 45 cycles; ntc, no template control.

Figure 3. Differences in basal and Ang-II stimulated aldosterone levels and adrenal CYP11B2 gene expression in mice by genotype and sex.

(A) Basal plasma aldosterone levels in male and female KO (KCNJ5^−/−), heterozygous (KCNJ5^+/−) and WT (KCNJ5^+/+) mice (n=7). *P<0.05 compared with male WT, **P<0.05 compared with female WT. (B) Levels of Cyp11B2 expression in WT compared with KO male and female mice (n=4). (C) Levels of plasma aldosterone before and 30 mins after Ang-II in male and female KO (KCNJ5^−/−) mice (n=4–7). *P<0.05 compared with basal, ^#P<0.05 male compared with female KO. Bars are mean ± S.E.M.

Figure 4. iSTAT blood and haematocrit measurements and body weight for WT (^+/+), heterozygous (^+/−), and KO (^−/−) mice by sex

The horizontal lines are mean ± S.E.M. for n=4–7 mice. *P<0.001 male compared with female.

Figure 5. Venn diagram representation of the differentially expressed genes (female compared with male) in adrenals from WT and KO (KCNJ5^−/−) mice

The top seven genes in both gene lists are identical.

Figure 6. Aldosterone production by cultured H295R adrenal cells exposed to PPAR agonists

(A) Aldosterone production from H295R cells treated with either control (0.1% DMSO) or 10 μ PPAR agonists: rosiglitazone (Rosi, PPARγ) or fenofibrate (Feno, PPARα) for 48 h. Cells were also treated for the final 24 h with 10 nM Ang-II or Ang-II with 10 nM losartan (Ang-II + L). Error bars represent S.E.M. of n=4. *P<0.05 compared with basal control, **P<0.05 compared with basal of same treatment, ^#P<0.05 compared with Ang-II of same treatment. (B) CYP11B2 gene expression in H295R cells treated for 24 h with either control (DMSO), 10 μM fenofibrate, 10 μM fenofibrate with GW6471 or 10 μM GW6471 alone. Error bars represent S.E.M. of n=4. *P<0.05 compared with control, ^#P<0.05 compared with Feno.

Figure 7. Effect of in vivo administration of fenofibrate on plasma aldosterone levels and adrenal CYP11B2 gene expression

(A) Effect of 7 or 14 days administration of fenofibrate (100 mg/kg PO) or vehicle (olive oil) on plasma aldosterone levels in female WT mice. *There is a significant treatment effect on two-way ANOVA (P=0.003). (B) Expression of CYP11B2 in the adrenals after 14 days of dosing with fenofibrate (Feno) or vehicle, *P<0.05 compared with control. Bars are mean ± S.E.M. for n=4.