Epigenetic modulation of the renal β-adrenergic–WNK4 pathway in salt-sensitive hypertension

Journal name:
Nature Medicine
Volume:
17,
Pages:
573–580
Year published:
DOI:
doi:10.1038/nm.2337
Received
Accepted
Published online
Corrected online
Corrected online

Abstract

How high salt intake increases blood pressure is a key question in the study of hypertension. Salt intake induces increased renal sympathetic activity resulting in sodium retention. However, the mechanisms underlying the sympathetic control of renal sodium excretion remain unclear. In this study, we found that β2-adrenergic receptor (β2AR) stimulation led to decreased transcription of the gene encoding WNK4, a regulator of sodium reabsorption. β2AR stimulation resulted in cyclic AMP-dependent inhibition of histone deacetylase-8 (HDAC8) activity and increased histone acetylation, leading to binding of the glucocorticoid receptor to a negative glucocorticoid−responsive element in the promoter region. In rat models of salt-sensitive hypertension and sympathetic overactivity, salt loading suppressed renal WNK4 expression, activated the Na+-Cl cotransporter and induced salt-dependent hypertension. These findings implicate the epigenetic modulation of WNK4 transcription in the development of salt-sensitive hypertension. The renal β2AR-WNK4 pathway may be a therapeutic target for salt-sensitive hypertension.

At a glance

Figures

  1. Effects of salt loading on blood pressure, renal WNK4 and NCC expression in norepinephrine (NE)-infused C57BL/6j, [beta]1AR-knockout ([beta]1-KO) and [beta]2AR-KO ([beta]2-KO) mice.
    Figure 1: Effects of salt loading on blood pressure, renal WNK4 and NCC expression in norepinephrine (NE)-infused C57BL/6j, β1AR-knockout (β1-KO) and β2AR-KO (β2-KO) mice.

    (a) Recordings of mean arterial pressure in NE-infused mice on normal-salt (0.3%) diet for 3 d, high-salt (8%) diet for 3 d and low-salt (0.05%) diet for 3 d. Closed circles, averages of mean arterial pressure measured every 30 min by radiotelemetry. (b) mRNA (left) and protein (right) amounts of renal WNK4 in control (Con), NE- or NE plus propranolol (Pro)-treated mice. The ratio of WNK4 to β-actin mRNA or protein relative to that in control mice is shown. (c) Effects of salt loading on mean arterial pressure in isoproterenol (Iso)-infused β1-KO and β2-KO mice. (d) Effects of salt loading (HS) and NE on renal WNK4 mRNA levels in WT, β1-KO and β2-KO mice. (e) Amounts of NCC protein and phosphorylated NCC protein (p-NCC) in control, NE- or NE plus propranolol (Pro)-treated mice. The same samples were used in b, and the actin blot shown is the same in b.Quantitative data were normalized using actin as a loading control. (f) Immunofluorescent micrographs of NCC in the kidney for each of the three groups of mice. Nuclei are stained by DAPI. Data are means ± s.e.m.; n = 4–6 for each group of mice. *P < 0.01 versus WT or control; #P < 0.01 versus NE or normal salt. NS, not significant.

  2. Effects of isoproterenol (Iso), hydrochlorothiazide (HCTZ) and ICI118551, a [beta]2-specific antagonist, on UNaV, plasma volume (PV) and blood pressure in rats fed a high-salt diet (HS).
    Figure 2: Effects of isoproterenol (Iso), hydrochlorothiazide (HCTZ) and ICI118551, a β2-specific antagonist, on UNaV, plasma volume (PV) and blood pressure in rats fed a high-salt diet (HS).

    (a) Left, effects of acute NCC blockade with intravenous injection of HCTZ (with HCTZ) and ICI 118551on FENa. Right, effect of acute NCC blockade with HCTZ injection on UNaV. (b) Left, effects of chronic infusion of Iso and HCTZ on daily UNaV and PV in HS rats. Right, percentage change in PV during the Iso infusion, as estimated by changes in hematocrit, in HS + Iso rats and in HS + Iso + HCTZ rats (P < 0.01, paired test). (c) Effects of HCTZ on salt-induced elevation of mean arterial pressure in Iso-infused rats. (d) Renal function curve of rats fed normal-salt and high-salt diet and treated or not with Iso or with Iso + HCTZ. Renal function curve was estimated by mean ± s.e.m. of blood pressure and daily UNaV. Data are means ± s.e.m.; n = 4–6 rats for each group. *P < 0.01 versus HS; #P < 0.05 for comparisons indicated in the figure.

  3. Role of glucocorticoid receptor (GR) in Iso-induced WNK4 inhibition and blood pressure elevation.
    Figure 3: Role of glucocorticoid receptor (GR) in Iso-induced WNK4 inhibition and blood pressure elevation.

    (a) Effects of Iso, dexamethasone (Dex), RU486 and H89 on WNK4 mRNA levels in mDCT cells in charcoal-stripped medium. (b) Effects of Iso, Dex and H89 on WNK4 transcription as measured by a luciferase assay in mDCT cells transfected with GR siRNA. Percentage changes of WNK4 transcription compared with control were calculated. (c) ChIP assay for GR binding to the promoter region of WNK4 containing nGRE in mDCT cells. Cells were treated or not with Dex, Iso or H89 as indicated. Bottom, relative GR binding to nGRE (PCR product / input). (Input and IgG results are shown in Supplementary Fig. 18b.) (d) Effects of NE and Dex on renal WNK4 mRNA levels in adrenalectomized mice (Adx). (e) Effects of a high-salt diet on renal WNK4 mRNA levels in Iso-infused WT and GR-knockout (GR-KO) mice. (f) Effects of a high-salt diet on average of mean arterial pressure measured every 30 min by radiotelemetry in Iso-infused WT and GR-KO mice. Data are means ± s.e.m.; in vitro (ad) n = 5 or 6 experiments in mDCT cells for each group; in vivo experiments (e,f) n = 5 or 6 mice for each group. *P < 0.01 versus control or normal salt; #P < 0.01; **P < 0.05 (versus WT + NS in e).

  4. Effects of Iso on nuclear GR protein and histone modulation of WNK4 transcription.
    Figure 4: Effects of Iso on nuclear GR protein and histone modulation of WNK4 transcription.

    (a) Top, western blotting to detect nuclear GR protein at 60 and 180 min after the indicated treatments in mDCT cells. Bottom, nuclear GR (n-GR) / total GR (t-GR) after 180 min of the indicated treatments. (b) Effects of Iso, Dex and H89 on acetylation of histone 3 (Ac-H3) and histone 4 (Ac-H4). Acetylated histone to β-actin ratio was calculated. (c) Quantitative immunoblot analysis of acetylation sites of histones H3 and H4. The level of acetylation of the indicated histone sites relative to that of the control group was calculated. (d) Chromatin immunoprecipitation (ChIP) assay for the presence of acetylated H3 and H4 in the promoter region of WNK4 containing nGRE in mDCT cells. The ratios of treatment to control PCR product quantities were calculated as relative ChIP. (e) WNK4 transcription as measured by a luciferase assay using deletion mutants containing 300 bp (–300) or 400 bp (–400) of the WNK4 promoter region in mDCT cells with the indicated treatments. TSA, trichostatin A. Percentage changes of WNK4 promoter transcription as compared to control were calculated. Data are means ± s.e.m.; n = 4–6 experiments in mDCT cells for each group. *P < 0.01 versus Con; #P < 0.01.

  5. Effects of Iso treatment on HDAC8 activity and H3 and H4 acetylation in the WNK4 promoter region.
    Figure 5: Effects of Iso treatment on HDAC8 activity and H3 and H4 acetylation in the WNK4 promoter region.

    (a) Effects of Iso and Dex on total HDAC (tHDAC) and Ser39 phosphorylated HDAC8 (pHDAC8) levels in mDCT cells. The amounts of phosphorylated HDAC8 as compared to control were calculated. (b) Effects of Iso and Dex on HDAC8 activity in mDCT cells transfected with plasmids expressing HDAC8 or HDAC8 S39A. (c) HDAC8 was immunoprecipitated (IP) from extracts of mDCT cells with the indicated treatments and immunoblots (IB) were performed to determine H3 and H4 binding to HDAC8. The cells had been transfected with plasmids expressing WT HDAC8 (left) or HDAC8 S39A (right). Relative binding of HDAC8 to H3 and H4 compared to control group were calculated. (d) ChIP assay for the presence of acetylated H3 (left) and acetylated H4 (right) in the promoter region of WNK4 containing nGRE in mDCT cells. The cells had been transfected with plasmids expressing WT HDAC or HDAC8 S39A and were treated or not with Dex, ISO or H89 as indicated. (Input and IgG results are shown in Supplementary Fig. 18b.) (e) ChIP assay for the binding of GR to the promoter region of WNK4 containing nGRE in mDCT cells which had been transfected with plasmids expressing WT HDAC8 or HDAC8 S39A and treated as indicated. (f) WNK4 transcription as measured by a luciferase assay in mDCT cells which had been transfected with plasmids expressing WT HDAC8 or HDAC8 S39A or with HDAC8 siRNA and treated as indicated, as compared to control group. Data are means ± s.e.m.; mDCT cells: n = 5 or 6 experiments in mDCT cells for each group. *P < 0.01 versus Con; #P < 0.01.

  6. Renal NE turnover, renal WNK4 expression and mean arterial pressure in DOCA-salt rats and salt-loaded Dahl-S and Dahl-R rats.
    Figure 6: Renal NE turnover, renal WNK4 expression and mean arterial pressure in DOCA-salt rats and salt-loaded Dahl-S and Dahl-R rats.

    (a) Renal NE turnover in SD rats fed a normal-salt or high-salt (HS) diet and DOCA-salt rats (left) and in salt-loaded Dahl-R and Dahl-S rats (right). Renal NE content was measured before and 6 h after addition of α-methyl-tyrosine. (b) Effects of salt loading and renal denervation (DNx) on renal WNK4 mRNA levels in SD rats. (c,d) In DOCA-salt rats, effect of DNx and addition of RU486 or Pro on renal WNK4 mRNA levels (c) and mean arterial pressure (d). (e) Effect of salt loading on WNK4 mRNA levels (left) and mean arterial pressure (right) 2 and 4 weeks later in Dahl-S rats and Dahl-R rats. (f) Effects of renal denervation, eplerenone, prazosin or propranolol treatment on renal WNK4 mRNA levels (left) and blood pressure (right) in salt-loaded Dahl-S rats. (g) Cartoon of a hypothetical mechanism for the development of salt-sensitive hypertension. Salt-induced renal sympathetic overactivity induces renal WNK4 downregulation and leads to sodium retention through NCC activation, thus resulting in salt-sensitive hypertension. Sympathetic overactivity leads to β2AR stimulation, HDAC8 phosphorylation and increased histone acetylation in the WNK4 promoter region, resulting in transcriptional modulation dependent on GR binding to nGREs in this region. Data are means ± s.e.m.; n = 4–6 rats in for each group. *P < 0.01 versus NS; #P < 0.01 versus HS; **P < 0.05 versus DOCA-HS.

Change history

Corrected online 04 August 2011

In the version of this article initially published, the authors made several inadvertent errors during manuscript preparation. In Figure 3f the trace for WT mice was incorrect, and in Figure 4a the bands shown for 'Total GR' were incorrect. These errors did not affect the quantification of band intensities shown in Figure 4a and did not affect any of the conclusions of the article. The errors have been corrected in the HTML and PDF versions of the article.
Corrected online 05 April 2012

In the version of this article initially published, the image of the actin bands shown in Supplementary Figure 2b was mistakenly rotated 180 degrees. The image has been replaced with the bands in their correct orientation, and the densitometry shown for this blot has been recalculated, which does not affect the conclusions. The figure legends for Figure 1e and Supplementary Figure 2b have also been edited to indicate that the same kidney samples were used for the blots in Figure 1b,e and Supplementary Figure 2b and that the actin bands shown for these blots are identical.

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Author information

Affiliations

  1. Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

    • ShengYu Mu,
    • Tatsuo Shimosawa,
    • Sayoko Ogura,
    • Hong Wang,
    • Yuzaburo Uetake,
    • Fumiko Kawakami-Mori,
    • Takeshi Marumo &
    • Toshiro Fujita
  2. Department of Clinical Laboratory, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

    • Tatsuo Shimosawa &
    • Yutaka Yatomi
  3. West Haven Veterans Affairs Medical Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • David S Geller
  4. Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

    • Hirotoshi Tanaka

Contributions

S.Y.M. carried out both in vitro and in vivo experiments and wrote the manuscript during a PhD course under the direction of T.F. at the University of Tokyo; T.S. carried out in vivo experiments and conducted experiments; S.O., H.W., Y.U., F.K.-M., Y.Y. and T.M. helped with experimental procedures and contributed to data discussion; D.S.G. generated distal nephron-specific glucocorticoid receptor–knockout mice and H.T. provided glucocorticoid receptor plasmids and contributed to data discussion. T.F. designed and directed the project and wrote the manuscript.

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The authors declare no competing financial interests.

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