Endogenous Angiotensin II‐induced p44/42 Mitogen‐Activated Protein Kinase Activation Mediates Sodium Appetite but not Thirst or Neurohypophysial Secretion in Male Rats

The renin–angiotensin–aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present study used an experimental model of endogenous AngII to examine the role of p44/42 mitogen‐activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), comprising a diuretic and an angiotensin‐converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviours. Central injection of U0126 markedly attenuated furo/cap‐induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap‐ or exogenous AngII‐induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII‐induced sodium appetite but not thirst or neurohypophysial secretion. This result may allow for the discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, at the same time as leaving thirst and neurohypophysial hormone secretion undisturbed

Endogenous Angiotensin II‐induced p44/42 Mitogen‐Activated Protein Kinase Activation Mediates Sodium Appetite but not Thirst or Neurohypophysial Secretion in Male Rats

The renin–angiotensin–aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present study used an experimental model of endogenous AngII to examine the role of p44/42 mitogen‐activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), comprising a diuretic and an angiotensin‐converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviours. Central injection of U0126 markedly attenuated furo/cap‐induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap‐ or exogenous AngII‐induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII‐induced sodium appetite but not thirst or neurohypophysial secretion. This result may allow for the discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, at the same time as leaving thirst and neurohypophysial hormone secretion undisturbed

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Robust antibody responses are induced by all vaccination regimens.

<p>Serum levels of total IgG (green triangles), IgG1 (black circles) and IgG2a (red circles) were measured at weeks 5, 11 and 15. A) Mice were primed at weeks 0 and 3 with Env-CN54 gp140 with alum alone, or a medium (“med”) or high concentration of IC31 with or without alum, and boosted at week 6 and 9 with NYVAC-CN54. B) Mice were primed at weeks 0 and 3 with NYVAC-CN54 and boosted at week 6 and 9 with Env-CN54 gp140 protein in combination with the adjuvants as indicated. Total IgG could not be detected in the control mice. The graphs show the antibody titers detected by ELISA at weeks 5, 11, and 15. Bars correspond to the medians. *p<0.05.</p

The memory T-cell pool is enhanced by Env/IC31 prime.

<p>A) At week 11, the numbers of CD8+ memory precursor cells (IFN-γ+ and/or TNF-α+ CD8+ KLRG1-low CD127+) in the spleen were determined for mice that were primed with Env-CN54 gp140 with various adjuvants as indicated and boosted with NYVAC-CN54. The graph shows the results obtained after <i>in vitro</i> stimulation with peptide pool 1. B) At week 15, memory T-cell numbers were assessed in the spleen. Central memory T cells were defined as CD8+ T cells secreting IFN-γ and/or TNF-α with high expression of CD62L. The graph depicts central memory cell numbers induced by the various vaccination regimens following <i>in vitro</i> re-stimulation with peptide pool 1. C) Proliferation of spleen CD8+ T cells from week 15 was determined by CFSE dilution. The graph shows the frequency of proliferating CD8+ T cells following <i>in vitro</i> stimulation with peptide pool 1 for 3 days. D) Effector memory T cells from week 15 were defined as CD8+ T cells secreting IFN-γ and/or TNF-α, expressing low levels of CD62L. The graph shows the values obtained after <i>in vitro</i> stimulation with peptide pool 1. Bars indicate the median value. *p<0.05 and **p<0.01. Each data point corresponds to a single mouse in the experimental group.</p

Schematic representation of the vaccination protocol.

<p>Three to five mice for each timepoint were injected at weeks 0 and 3 with the prime and at weeks 6 and 9 with the boost. Mice were either injected with saline (negative control, three mice per group), with protein plus alum alone, with protein plus a medium or high dose of IC31 alone, with protein plus alum and a medium or high dose of IC31, or with NYVAC-CN54 (five mice per each group per timepoint). At week 5, 11 or 15, a subset of mice were sacrificed and antibody and T-cell responses were analyzed <i>ex vivo</i>.</p

Durable Env-CN54 specific CD4+ T-cell responses are detected following vaccination when Env/alum/IC31 is used as a prime.

<p>(A–C) Mice were primed with Env-CN54 gp140 in combination with either alum alone or a medium (“med”) or high concentration of IC31 with or without alum and then boosted with NYVAC-CN54 at three week intervals. A) Frequencies of activated CD4+ T cells as indicated by low CD62L expression at weeks 5, 11 and 15. Each data point corresponds to a single mouse in the experimental group. B) Percentage of CD4 T cells secreting cytokines (TNF-α and IFN-γ) upon stimulation with four peptide pools covering the full length of Env-CN54 gp140, as determined by ICS. C) Kinetic analysis of the frequencies of cytokine secreting CD4+ T cells following vaccination. Data points correspond to the medians of the frequencies obtained by stimulation with peptide pool 1. D–E) Mice were primed with NYVAC-CN54 and boosted with Env-CN54 gp140 in combination with adjuvants as listed. (D) Frequencies of activated CD4+ T cells as indicated by low CD62L expression at weeks 11 and 15. Each data point corresponds to a single mouse in the experimental group. (E) Percentage of CD4+ T cells secreting cytokines at weeks 11 and 15. For B and E, data points following stimulation with peptide pool 1 (red circles), pool 2 (green squares), pool 3 (green triangles) and pool 4 (black triangles) are distinguished, while the dotted line corresponds to the average obtained upon DMSO stimulation. Bars correspond to median values (medians for peptide pool 1 stimulation only in B and E). *p<0.05.</p

Detection of Env-CN54 gp140 specific CD8+ T cells in the GALT of vaccinated mice.

<p>The frequencies of CD8+ T cells secreting cytokines in response to CN54 peptide pools were evaluated in the mesenteric lymph nodes (MLN) of vaccinated mice at week 11 (A) and at week 15 (B). Graphs show the median CD8+ T-cell responses from the MLN (white bars) and from spleen (black bars) following stimulation with peptide pool 1. C) Effector memory T-cell numbers within the MLN were determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042163#pone-0042163-g005" target="_blank">Figure 5</a>. Each data point corresponds to a single mouse in the experimental group.</p