Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 28, 2015). doi:10.1152/ajpregu.00444.2014
1
Title: A comparison of physiological and transcriptome responses to water
2
deprivation and salt loading in the rat supraoptic nucleus
3
Running title: Osmotic stimulation of the SON
4
Authors: Michael P Greenwood1, Andre S Mecawi2,3, See Ziau Hoe2, Mohd
5
Rais Mustafa4, Kory R Johnson5, Ghada A. Al-Mahmoud6, Lucila LK Elias7,
6
Julian FR Paton8, Jose Antunes-Rodrigues7, Harold Gainer9, David
7
Murphy*1,2, Charles CT Hindmarch*§1,2
8 9 10 11 12 13 14
* Equal senior authors § Corresponding author:
[email protected]
15 16
2
17 18
3
19 20
1
School of Clinical Sciences, Dorothy Hodgkin Building, University of Bristol, Bristol, England, BS1 3NY Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 50603 Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, Seropedica, Brazil 4
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 50603
21 22
5
23 24
6
25 26
7
27 28
8
29 30
9
31 32 33 34 35 36 37 38
NINDS/NIH, Clinical Center (Building 10), Office 5S223, 9000 Rockville Pike, Bethesda, MD, 20892 Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, Al Tarfa, Doha 2713, Qatar Department of Physiology, School of Medicine of Ribeirao Preto, University of S. Paulo, Ribeirao Preto - 14049-900, Ribeirao Preto - SP - Brazil School of Physiology and Pharmacology, Medical Sciences Building, University Walk, Bristol BS8 1TD Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA Author contributions: CCTH, DM, and HG, designed the experiments. CCTH, MPG, and ASM analysed and interpreted the results. CCTH, MPG, ASM, HSZ, MRM performed the experiments with help from GA-M. CCTH performed and analysed the transcriptomics. CCTH and DM wrote the manuscript with editorial input from all authors.
39
Copyright © 2015 by the American Physiological Society.
40
Abstract
41
Salt loading (SL) and water deprivation (WD) are experimental challenges that
42
are often used to study the osmotic circuitry of the brain. Central to this circuit
43
is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for
44
the biosynthesis of the hormones, vasopressin (AVP) and oxytocin (OXT),
45
and their transport to terminals that reside in the posterior lobe of the pituitary.
46
Upon osmotic challenge evoked by a change in blood volume or osmolality,
47
the SON undergoes a function related plasticity that creates an environment
48
that allows for an appropriate hormone response. Here, we have described
49
the impact of SL and WD compared to euhydrated (EU) controls in terms of
50
drinking and eating behaviour, body weight and recorded physiological data
51
including circulating hormone data and plasma and urine osmolality. We have
52
also used microarrays to profile the transcriptome of the SON following SL
53
and re-mined data from the SON that describes the transcriptome response to
54
WD. From a list of 2,783 commonly regulated transcripts, we selected 20
55
genes for validation by qPCR. All of the 9 genes that have already been
56
described as expressed or regulated in the SON by osmotic stimuli were
57
confirmed in our models. Of the 11 novel genes, 5 were successfully validated
58
while 6 were false discoveries.
59
Key words: Transcriptome, Supraoptic nucleus, Water restriction, Salt
60
load, neuroendocrine
61 62 63 64
65
Introduction:
66
Terrestrial life requires that the osmolality of the extracellular fluid (ECF) is
67
strictly controlled. Increased ECF osmolality results in water leaving the cell,
68
reducing intracellular fluid (ICF) volume whilst increasing ICF osmolality,
69
which will compromise the metabolic processes necessary for life. Chronic
70
increases in ECF osmolality can be brought about experimentally by a high
71
intake of salt (salt loading, SL), or by water deprivation (WD) (5). In the
72
absence of drinking fluid (WD), extracellular and intracellular fluid volumes
73
decrease, as water and sodium are inevitably lost in sweat and urine leading
74
to hypovolemia and, as a consequence of dehydration-induced natriuresis,
75
sodium depletion (14, 35). This depletion of sodium means that WD animals
76
also display enhanced salt appetite (14). In contrast, SL increases body
77
sodium content, causing an increase in ECF volume, but a decrease in the
78
volume of the ICF (35).
79
Angiotensin II (ANG II), atrial natriuretic peptide (ANP), arginine vasopressin
80
(AVP) and oxytocin (OXT) are the main hormones involved in the control of
81
hydromineral homeostasis in mammals (5). ANP is synthesized and secreted
82
into the bloodstream in response to stretching of the right atrial muscle cells
83
by increased blood volume. Once in the bloodstream, ANP has a potent
84
natriuretic effect, acting on the distal convoluted tubule of the nephron to
85
inhibit sodium reabsorption (13, 44). ANP is an important indicator of blood
86
volume. AVP and OXT release are stimulated by hypovolemia, hypertonicity
87
and hypernatremia, among other stimuli (36). Circulating levels of AVP
88
correlate with plasma osmolality (4), and thirst perception (3, 42) and serves
89
to increase permeability of the collecting duct in the kidney allowing water to
90
be conserved and urine to be concentrated. In relation to osmotic control,
91
OXT is a natriuretic hormone that is elevated early in response to osmotic
92
challenge, in order to reduce sodium appetite and influence sodium balance
93
through increased excretion (59). ANG II exerts several effects on
94
hydromineral balance, including the regulation of thirst and sodium appetite,
95
AVP secretion and renal water and sodium reabsorption (4, 37). The enzyme
96
renin is responsible for the cleavage of angiotensinogen into angiotensin I,
97
which in turn is cleaved into ANGII. Renin is stimulated by reducing the Na+
98
content that reaches the distal convoluted tubules of the nephron (31). The
99
subfornical organ (SFO) expresses the angiotensin receptor 1A (At1A; (22))
100
and the expression of this receptor changes following osmotic stimulation
101
through water deprivation (7, 11). Moreover, the SFO directly projects to
102
vasopressinergic magnocellular neurons of the supraoptic nucleus (SON;
103
(61)).
104
Evolution has thus ensured that cells are protected from fluctuations in
105
the osmolality of the extracellular environment. Brain mechanisms involved in
106
the regulation of osmolality are centred on the central osmoregulatory circuit.
107
Osmosensitive neurons of the circumventricular SFO communicate chronic
108
changes in plasma osmolality through direct projections to the magnocellular
109
neurons (MCNs) of the hypothalamic supraoptic (SON) and paraventricular
110
(PVN) nuclei, thus modulating the synthesis of AVP and OXT, and their
111
transport to, and release from, the posterior lobe of the pituitary (26, 61).
112
When ECF osmolality rises, the SON responds with an increase in AVP
113
synthesis and release that is accompanied by change in soma size
114
(hypertrophy), the result of the intracellular organelle development required to
115
respond to the stimulus in terms of hormone production. The SON
116
experiences a function related remodelling that is necessary for the optimal
117
facilitation of hormone production and delivery (20, 50, 54). These
118
morphological changes are accompanied by biochemical events such as a
119
strong activation of the cAMP pathway (10) and transcriptional events that
120
extend beyond simple AVP and OXT biogenesis (32, 46). We have previously
121
shown that WD in both male and female rats is capable of modulating gene
122
expression in the SON (23, 45) and that this modulation is a global
123
transcriptome response (24). We have also recently demonstrated that many
124
of the genes that are regulated in the SON in response to WD are also altered
125
by lactation (45).
126
While
salt-loading
we
deprivation demonstrate
are here
often that
seen
as
experimentally
128
physiological differences exist between them at the level of drinking, feeding,
129
weight loss, hormonal response and responsiveness to water repletion at the
130
end of the challenge. We wanted to ascertain whether these differences were
131
managed at the level of the SON and have therefore profiled the
132
transcriptome of this tissue following 7-days salt-loading and compared it to
133
our previous transcriptome data from the water deprived SON (23) in order to
134
assess whether those genes that are similarly or differently regulated are
135
relevant to the physiological changes we see.
137
stimuli,
water
127
136
similar
and
profound
138
Methods
139
Animals: Adult, 10-12 weeks old male Sprague-Dawley (Harlan Sera-lab,
140
Loughborough, UK) and Wistar rats (Central Animal Facility University of São
141
Paulo, Ribeirão Preto, Brazil) were maintained in standardised temperature
142
(22±1°C), humidity (50±5%) and diurnal conditions (UK: 10 hours light, 14
143
hours dark; lights on at 0700, BR: 12 hour light, 12 hours dark). Water
144
deprivation (WD) involved complete fluid deprivation for up to 3 days, whereas
145
salt-loading (SL) involved the replacement of drinking water with 2% (w/v in
146
tap water) NaCl for up to 7 days; all animals had access to food (standard
147
laboratory rat chow) ad libitum throughout the experiment. The euhydrated
148
(EU) control group of animals had access to both food and normal drinking
149
water for the duration of the experiment. Rats were sacrificed (between the
150
hours of 0900 and 1300) and the tissue isolated and processed as described
151
below. All experiments were carried out under the licencing arrangements of
152
the UK Animals (Scientific Procedures) Act (1986) and Brazilian legislation
153
(law number 11794, October 8, 2008) with both local ethics committee
154
approval.
155
Metabolic cage Experiments
156
To enable precise daily measures of fluid intake, food intake and urine output
157
animals (n=6 per group) were individually housed in metabolic cages
158
(Techniplast, Italy). Fluid intake, food intake and urine output were recorded
159
after 3d acclimatisation (control measure) and during 3d WD and 7d SL.
160
Animals were killed by stunning (which involved a blow to the head), followed
161
by decapitation prior to trunk blood collection.
Plasma and urine osmolality
162
was determined by freezing point depression using a Roebling micro-
163
osmometer (Camlab).
164
Physiological assessment and water repletion
165
In total, 20 rats were single housed and randomly assigned to one of three
166
groups – 7d SL (n=7), 3d WD (n=7) or EU controls (n=6). Cumulative fluid
167
consumption by the EU and SL animals was measured by weight each day
168
(between 10.00-11.00 am) for 7 days. Following 7 days SL and 3 days WD,
169
animals were given free access to tap-water and fluid intake was recorded
170
after 15 minutes, 30 minutes, 45 minutes and then hourly until 6 hours and
171
then again at 24 hours at which point the animal was sacrificed. Cumulative
172
water consumption per animal was normalised for spillage (calculated at 3ml)
173
and calculated as mL/Kg based on starting body weight.
174
Hormone assessment
175
In total, 21 rats were grouped housed and randomly assigned to one of three
176
groups – SL (n=7), 2 days WD (n=7) or EU controls (n=7). Animals were
177
decapitated and blood was collected from the trunk into chilled tubes
178
containing heparin for AVP and OXT or peptidase inhibitors for ANG II and
179
ANP levels. Plasma was obtained after centrifugation (20 min, 1600g, 4°C)
180
and stored at −20°C until specific extraction and radioimmunoassay
181
procedures. The specific antibody for ANG II radioimmunoassay was obtained
182
from Peninsula (ANG II, T4007; San Carlos, CA); and for AVP and OT RIA
183
were donated by W.K. Samson (University of St. Luis, Mo, USA) and for ANP
184
was donated by Jolanta Gutkowska (Univ. of Montreal, Canada). The
185
radioimmunoassay sensitivity and intra- and interassay coefficients of
186
variation were 0.5 pg/ml, 8.7–11.2% for ANG II; 0.7 pg/ml, 4.1–7.8% for ANP;
187
0.1 pg/mL, 4.5–4.9% for AVP, 0.1 pg/mL, 5.1–8.8% for OT.
188
Tissue collection and RNA extraction for Microarray
189
Rats were stunned (which involves a blow to the head) then decapitated with
190
a small animal guillotine (Harvard Apparatus, Holliston, MA, USA). The brain
191
was rapidly removed from the cranium and placed in an ice-cold brain matrix
192
(ASI Instruments, Warren, MI, USA). One section of approximately 1mm
193
thickness was taken and the SON carefully dissected on a bed of ice under a
194
dissecting microscope (Leica, Germany). After isolation, the samples were
195
immediately placed in Eppendorf tubes containing RNAlater® (Ambion,
196
Huntingdon, UK). Tissue was mechanically homogenised in QIAzol Lysis
197
Reagent (QIAgen, Crawley, UK) and the aqueous phase removed after
198
centrifugation through a Phase Lock Gel column (Eppendorf, Cambridge, UK).
199
Total RNA was purified with RNeasy Micro Kit MinElute Spin Columns
200
(Qiagen, Crawley, UK) and eluted into 14μl of RNase free water. Following
201
quality control confirmation, total-RNA was amplified using the Affymetrix IVT
202
Express kit, hybridized to Affymetrix Rat GeneChip 230 2.0 microarrays
203
according to manufacturer’s instructions (Affymetrix). Arrays were scanned
204
using the GeneChip Scanner 3000 (Affymetrix). All data was analysed in
205
Genespring GX12 (Agilent); .CEL files from the SL and the EU arrays (n=5)
206
were uploaded subject to quartile RMA normalization where the median was
207
used for baseline before statistical differences between the two conditions
208
were determined using an unpaired t-test for unequal variance (Welch) with a
209
Benjamini Hotchberg multiple test correction applied to maintain a corrected
210
p-value of
