09 August 2022
AperTO - Archivio Istituzionale Open Access dell'Università di Torino
Original Citation:
Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles
Published version:
DOI:10.1161/HYPERTENSIONAHA.120.15998
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Effect of dietary sodium modulation on pig adrenal steroidogenesis and transcriptome profiles
Twinkle Vohra
1*
, Elisabeth Kemter
2*
, Na Sun
3
, Britta Dobenecker
4
, Arne Hinrichs
2
, Jacopo Burrello
5
,
Elise P. Gomez-Sanchez
6
, Celso E. Gomez-Sanchez
7
, Jun Wang
3
, Isabella-Sabrina Kinker
1
, Daniel
Teupser
8
, Konrad Fischer
9
, Angelika Schnieke
9
, Mirko Peitzsch
10
, Graeme Eisenhofer
10,11
, Axel
Walch
3
, Martin Reincke
1
, Eckhard Wolf
2
†
, Tracy Ann Williams
1,5
†
1) Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ludwig-Maximilians-Universität
München, Munich, Germany (T Vohra, I-S Kinker, M Reincke, TA Williams)
2) Chair for Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary
Sciences, Ludwig-Maximilians-Universität München, Munich, Germany (E Kemter, A Hinrichs, E Wolf)
3) Research Unit Analytical Pathology, German Research Center for Environmental Health, Helmholtz
Zentrum München, Neuherberg, Germany (N Sun, A Walch)
4) Chair of Animal Nutrition and Dietetics, Department of Veterinary Sciences, Ludwig-Maximilians-
Universität München, Oberschleißheim, Germany (B Dobenecker)
5) Division of Internal Medicine and Hypertension, Department of Medical Sciences, University of Turin,
Turin, Italy (J Burrello, TA Williams)
6) Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, Jackson,
MS, USA (EP Gomez-Sanchez)
7) Endocrine Division, G.V. (Sonny) Montgomery VA Medical Center, and Department of Pharmacology and
Toxicology and Medicine, University of Mississippi Medical Center, Jackson, MS, USA (CE Gomez-Sanchez)
8) Institute of Laboratory Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich,
Germany (D Teupser)
9) School of Life Sciences Weihenstephan, Technical University Munich, Freising, Germany (K Fischer, A
Schnieke)
10) Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus,
Technische Universität Dresden, Dresden, Germany (G Eisenhofer, M Peitzsch)
11) Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden,
Dresden, Germany (G Eisenhofer)
*
These authors contributed equally and should be considered joint first authors
†
These authors contributed equally and should be considered joint last authors
Corresponding author: Tracy Ann Williams PhD, Medizinische Klinik und Poliklinik IV, Klinikum der
Universität München, LMU München, Ziemssenstr. 1, D-80336 München, Germany. Tel: +49 89
4400 52941; Fax: +49 89 4400 54428; Email: Tracy.Williams@med.uni-muenchen.de
Total words: 2700 excluding references, 1 table, 4 figures;
online-only supplement with 6 tables, 3 figures
Running title: Pig adrenal response to sodium restriction
1
Abstract
1
Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone
2
overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied
3
in rodents despite differences in adrenal physiology with humans. We therefore investigated pig
4
adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and
5
zona fasciculata (zF) in response to activation of the renin-angiotensin-aldosterone system by
6
dietary sodium restriction. Six-week-old pigs were fed a low or high sodium diet for 14 days (3 pigs
7
per group, 0.4g sodium/kg feed versus 6.8g sodium/kg). Plasma aldosterone concentrations
8
displayed a 43-fold increase (p=0.011) after 14-days of sodium restriction (day 14 versus day 0).
9
Low dietary sodium caused a 2-fold increase in thickness of the zG (p<0.001) and an almost 3-fold
10
upregulation of CYP11B (cytochrome P450 11B1) (p<0.05) compared with high dietary sodium.
11
Strong immunostaining of the KCNJ5 potassium channel, which is frequently mutated in primary
12
aldosteronism, was demonstrated in the zG. mRNA-seq transcriptome analysis identified
13
significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system
14
in the zG (n= 1,172) and zF (n= 280). These genes included many with a known role in the
15
regulation of aldosterone synthesis and adrenal function. The most highly enriched biological
16
pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle and
17
potassium channels. This study provides mechanistic insights into the physiology and
18
pathophysiology of aldosterone production in a species closely related to humans and shows the
19
suitability of pigs as a translational animal model for human adrenal steroidogenesis.
20
Key words: aldosterone; cortisol; hyperaldosteronism; adrenal cortex; sodium restriction;
steroidogenesis; hypertension
2
Introduction
21
The mineralocorticoid hormone aldosterone is synthesized in zona glomerulosa (zG) cells of the
22
adrenal cortex and stimulates sodium reabsorption in epithelial cells of the kidney distal tubule
23
and colon for the maintenance of blood volume and blood pressure. The main physiological
24
regulators of aldosterone production are the renin-angiotensin-aldosterone system (RAAS) and
25
circulating potassium; although other factors are likely also involved.
1-3
Sodium depletion activates
26
the RAAS and causes expansion of the zG layer and an increase in aldosterone secretion.
4,5
27
Manipulation of dietary sodium in rats has been used as an approach to study the effects of RAAS
28
activation on zG gene expression to identify genes that function in the regulation of aldosterone
29
production.
5-7
30
31
Humans and commonly used experimental surrogates display distinct differences in adrenal
32
physiology. For example, the Cyp17a1 gene (encoding 17a-hydroxylase and 17,20-lyase) is not
33
expressed in the adrenal glands of laboratory rats and mice resulting in the production of
34
corticosterone as the major glucocorticoid, instead of cortisol as in humans. Another major
35
difference involves the absence of the zona reticularis (zR) and adrenal androgen synthesis in
36
these rodents.
8
Furthermore, potassium channels which function in the maintenance of zG cell
37
membrane potential show different gene expression profiles and patterns of immunostaining in
38
rat and human adrenals.
9,10
Notably, the rat adrenal does not express the inwardly rectifying
39
potassium channel KCNJ5 which is frequently mutated in primary aldosteronism.
9
Regulation of
40
KCNJ5 gene expression and channel activity modulate the zG membrane depolarization that
41
normally initiates aldosterone production in humans.
11
KCNJ5 mutations allow uncontrolled zG
42
membrane depolarization and cause aldosterone excess.
43
44
3
The adrenal cortex is divided into three morphologically distinct layers (zG, zF, and zR). In the
45
human adrenal, restricted expression of CYP11B2 (encoding aldosterone synthase) in the zG and
46
CYP11B1 (encoding 11β-hydroxylase) in the zF and zR sustains the functional zonation of
47
aldosterone biosynthesis in the zG and cortisol in the zF. These two CYP11B enzymes, with their
48
specific zonal distributions, are present in multiple species including mice, rats, hamsters, and
49
guinea pigs.
8
In contrast, others, such as pigs, cattle, sheep and dogs, express a single CYP11B
50
enzyme that performs the final steps of both aldosterone and cortisol biosynthesis
12
and, by way
51
of an unknown mechanism, their biosynthetic zonal specificity is maintained.
13,14
52
53
Large animals such as pigs are useful to model complex human diseases due to their comparable
54
anatomy and physiology to humans.
15-17
Such models provide an opportunity to screen for disease
55
biomarkers and test novel therapeutic strategies.
18,19
In this study, we evaluated the role of dietary
56
sodium manipulation on adrenal morphology, steroidogenesis and transcriptome profiles in 6-
57
week-old male pigs. Our objective was to identify the transcriptional response of the adrenal to
58
RAAS activation and determine the suitability of the pig as a translational animal model for human
59
adrenal steroidogenesis.
60
61
Methods
62
An expanded online methods section is available in the online supplementary file.
63
The authors declare that all supporting data are available within the article and its online
64
supplementary files. mRNA-seq data are publicly available and can be accessed at
65
https://github.com/MedIVLMUMunich/PigAdrenalRNAseq
66
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