Af17 deficiency increases sodium excretion and decreases blood pressure

Abstract

The putative transcription factor AF17 upregulates the transcription of the epithelial sodium channel (ENaC) genes, but whether AF17 modulates sodium homeostasis and BP is unknown. Here, we generated Af17-deficient mice to determine whether deletion of Af17 leads to sodium wasting and low BP. Compared with wild-type mice, Af17-deficient mice had lower BP (11 mmHg), higher urine volume, and increased sodium excretion despite mildly increased plasma concentrations of aldosterone. Deletion of Af17 led to increased dimethylation of histone H3 K79 and reduced ENaC function. The attenuated function of ENaC resulted from decreased ENaC mRNA and protein expression, fewer active channels, lower open probability, and reduced effective activity. In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and BP. Taken together, these data identify Af17 as a potential locus for the maintenance of sodium and BP homeostasis and suggest that a particular histone modification is directly linked to these processes. Af17-mediated regulation of BP is largely, but not exclusively, the result of modulating ENaC, suggesting it has potential as a therapeutic target for the control of BP.

Figure 1.

Af17^−/− mice fed the normal Na⁺ diet showed impaired renal function and decreased BP, despite mildly increased plasma aldo. Af17^+/+ (n = 34) and Af17^−/− (n = 37) mice fed the normal Na⁺ diet in metabolic cages were analyzed for the parameters as indicated. For BP measurement, n = 57 mice for Af17^+/+ and n = 65 mice for Af17^−/−. For all other parameters, n = 6 to 37 mice/group. For additional parameters, see Supplemental Figure S1. In all cases, *P < 0.05 versus Af17^+/+.

Figure 2.

Impairment and restoration of ENaC activity in Af17^−/− mice. (A) Representative current traces from cell-attached patches monitoring ENaC activity under conditions as indicated. Dashed lines indicate the respective current state with a c denoting the closed state. (B–D) Summary graph of ENaC Po (B), active channels within a patch (C), and effective ENaC activity (D). In all cases, *P < 0.05 versus Af17^+/+.

Figure 3.

Af17 regulates mRNA and protein expression of ENaC. (A) Real-time RT-qPCR for expression of ENaC genes in kidney of mice fed the normal Na⁺ diet, with β-actin as internal control. n = 3 mice/group. (B) Western blots for expression of proteins as indicated, with β-actin as internal control. For αENaC, the approximately 30-kD band was excluded in data analyses. me2K79 and me2K9: histone H3 dimethylated K79 and K9, respectively. n = 4 mice/group. In all cases, *P < 0.05 versus Af17^+/+.

Figure 4.

Af17 is expressed in aldo-sensitive distal nephron. (A and B) Sections of Af17^−/− kidney were stained with X-gal and an antibody against the principal cell marker Aqp2. Boxed regions in A and B were amplified in C and D, respectively. The arrowheads indicate cells apparently expressing Aqp2 and β-geo reporter driven by the Af17 promoter.

Figure 5.

Af17 deletion causes H3 K79 hypermethylation at the αENaC promoter in kidney. (A) Diagram of the αENaC promoter.^– (B and C) ChIP assay showing increased H3me2K79 associated with R0-R3, but not with Ra subregion of the αENaC promoter. Kidney chromatin was prepared from four WT and four mutant mice, pooled into two groups according to genotype, and analyzed by ChIP with the antibodies indicated, and followed by real-time qPCR with primers amplifying the subregions of αENaC promoter as shown in A. Relative H3me2K79 abundance was set to 1 in R0 from WT kidneys and was calculated accordingly for all other samples. *P < 0.05 versus Af17^+/+. n = 4 mice/group (B). Representative agarose gel analyses of the final qPCR products were shown to verify the specificity of qPCR for each sample (C).

Figure 6.

Aldo perfusion largely compensated for the loss of Af17 function in renal physiology. Twenty-four-hour urine volume, urinary electrolyte excretion, and BP were examined before (day 0) and after aldo perfusion as indicated. n = 10 to 11 mice/genotype. Dark bar: Af17^+/+. Gray bar: Af17^−/−. For additional parameters, see Supplemental Figure S2. In all cases, *P < 0.05 versus Af17^+/+.

Figure 7.

Af17^−/− and Af17^+/+ mice show similar renal physiology and BP in a time-dependent manner on the low Na⁺ diet. Af17^+/+ (solid line or dark bar) and Af17^−/− (dotted line or gray bar) mice were fed the low Na⁺ diet (0.02% Na⁺) in metabolic cages and analyzed for the parameters before (day 0) and after treatment at various time points as indicated. For BP measurement, n = 17 to 21 mice/group. For all other parameters, n = 4 to 14 for Af17^+/+ mice and n = 6 to 14 for Af17^−/− mice. For additional parameters, see Supplemental Figure S4. In all cases, *P < 0.05 versus Af17^+/+.

Figure 8.

On the high K⁺ diet, Af17^−/− and Af17^+/+ mice had similar renal physiology and BP on the high K⁺ diet. Af17^+/+ and Af17^−/− mice were fed the high K⁺ diet (6% K⁺) for up to 5 days in metabolic cages and analyzed for the parameters before (day 0) and after treatment as indicated. n = 8 to 26 for Af17^+/+ mice and n = 7 to 25 for Af17^−/− mice. For additional parameters, see Supplemental Figure S5. In all cases, *P < 0.05 versus Af17^+/+.

Figure 9.

Model for impairment and restoration of Na⁺ balance and BP in Af17^−/− mice. Under basal conditions such as the normal Na⁺ diet, deletion of Af17 leads to significantly increased H3 K79 methylation, and subsequently impaired ENaC activity, disturbance of Na⁺ balance, and decreased BP. The mildly increased plasma [aldo] is not sufficient to antagonize the effect of Af17 loss on H3 K79 methylation and thus fails to rescue the Af17^−/− phenotype. Under aldo excess such as aldo perfusion or the low Na⁺ diet dramatically increased aldo levels efficiently decrease H3 K79 methylation regardless of the presence or absence of Af17, leading to comparable levels of H3 K79 methylation and a renal physiology and BP phenotype indistinguishable between Af17^+/+ and Af17^−/− mice. For clarity, detailed mechanisms by which Af17 and aldo inhibit H3 K79 methylation are not shown. The dotted line indicates unknown mechanisms.