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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Hypertension. 2015 Apr 20;65(6):1341–1348. doi: 10.1161/HYPERTENSIONAHA.115.05377

Brain Endoplasmic Reticulum Stress Mechanistically Distinguishes the Saline-Intake and Hypertensive Response to DOCA-Salt

Fusakazu Jo 1, Hiromi Jo 1, Aline M Hilzendeger 1, Anthony P Thompson 2, Martin D Cassell 2, D Thomas Rutkowski 2, Robin L Davisson 4,5, Justin L Grobe 1,3, Curt D Sigmund 1,3
PMCID: PMC4433403  NIHMSID: NIHMS676148  PMID: 25895586

Abstract

Endoplasmic reticulum stress has become an important mechanism in hypertension. We examined the role of endoplasmic reticulum stress in mediating the increased saline intake and hypertensive effects in response to DOCA-salt. Intracerebroventricular delivery of the endoplasmic reticulum stress-reducing chemical chaperone Tauroursodeoxycholic acid did not affect the magnitude of hypertension, but markedly decreased saline intake in response to DOCA-salt. Increased saline intake returned after Tauroursodeoxycholic acid was terminated. Decreased saline intake was also observed after intracerebroventricular infusion of 4-phenylbutyrate, another chemical chaperone. Immunoreactivity to CHOP, a marker of irremediable endoplasmic reticulum stress, was increased in the subfornical organ and supraoptic nucleus of DOCA-salt mice, but the signal was absent in control and CHOP-deficient mice. Electron microscopy revealed abnormalities in endoplasmic reticulum structure (decrease in membrane length, swollen membranes, and decreased ribosome numbers) in the subfornical organ consistent with endoplasmic reticulum stress. Subfornical organ-targeted adenoviral delivery of GRP78, a resident endoplasmic reticulum chaperone, decreased DOCA-salt-induced saline intake. The increase in saline intake in response to DOCA-salt was blunted in CHOP-deficient mice, but these mice exhibited a normal hypertensive response. We conclude: 1) brain endoplasmic reticulum stress mediates the saline intake, but not blood pressure response to DOCA-salt, 2) DOCA-salt causes endoplasmic reticulum stress in the SFO which when attenuated by GRP78 blunts saline intake, and 3) CHOP may play a functional role in DOCA-salt-induced saline intake. The results suggest a mechanistic distinction between the importance of endoplasmic reticulum stress in mediating effects of DOCA-salt on saline intake and blood pressure.

Keywords: Endoplasmic reticulum stress, DOCA-salt hypertension, saline intake, SFO

Introduction

Treatment of mice with deoxycorticosterone acetate (DOCA)-salt is a model of hypertension attributed to low circulating but elevated brain renin-angiotensin system (RAS) activity. Implicating the mechanistic involvement of the brain RAS are studies showing that central injection of either angiotensin converting enzyme inhibitors or angiotensin II (Ang-II) type 1 receptor (AT1R) blockers into the brain either prevent or reverse DOCA-salt hypertension.1–3 DOCA-salt also causes an increased resting metabolic rate which can be significantly blunted by an AT1R blocker.4,5

The subfornical organ (SFO) is a circumventricular organ that plays an important role in the regulation of fluid balance, cardiovascular regulation, and energy metabolism.6,7 The SFO is a sensory brain structure which lies outside of the blood brain barrier and relays information about the constitution of cerebral spinal fluid to other parts of the central nervous system.8 The SFO expresses angiotensinogen, the substrate for Ang-II, and is also rich in AT1R.9,10 Ablation of angiotensinogen production in the SFO prevents the pressor and dipsogenic responses to increased Ang-II production in the brain.11,12 Moreover, ablation of AT1R in the SFO of DOCA-salt-treated mice attenuates the pressor and dipsogenic responses characteristic of this model of hypertension.13 These studies identify a critical role of AT1R in the SFO in mediating some of the cardiovascular and metabolic phenotypes associated with both Ang-II and DOCA-salt hypertension.

Recent evidence suggests that endoplasmic reticulum (ER) stress in the SFO contributes to the development of hypertension.14 Young et al. showed that intracerebroventricular infusion of the ER stress-inducing chemical thapsigargin caused hypertension. They further demonstrated that subcutaneous infusion of Ang-II at a dose which increases arterial pressure gradually over time caused ER stress in the SFO as detected by increased expression of ER stress biomarkers and ultrastructural abnormalities in the ER. The mechanistic association between brain ER stress and hypertension was demonstrated by showing that the hypertension induced by Ang-II could be prevented by treatment with a chemical chaperone or by SFO-directed expression of GRP78, the most abundant ER-resident chaperone.

The ER is a membranous organelle which has as its main function directing the synthesis and folding of proteins. Secretory proteins and resident proteins of the endomembrane system are translocated into the ER lumen, where they are folded, oxidized, and glycosylated prior to export to the Golgi for further modification and transit to their final destinations. Approximately one-third of the cellular proteome is folded in the ER, and an excess of ER client proteins or a perturbation that disrupts ER protein folding can induce ER stress and activate an adaptive signaling cascade known as the unfolded protein response (UPR). The UPR has been comprehensively reviewed.15,16 The UPR acts to restore ER homeostasis in several ways, including selective inhibition of protein synthesis, increased synthesis of ER-resident chaperones, and increased degradation of unfolded proteins. It is becoming clear that the UPR is activated by normal physiological stimuli, and thereby contributes to the maintenance of physiological homeostasis.17 In addition, a number of pathophysiological conditions, including diabetes, cancer, and cardiovascular disease, have been associated with excessive and/or persistent ER stress, which is thought to contribute to the disease phenotype.18–21 Persistent ER stress can contribute to pathophysiology both by inciting cell death and by leading to dysregulation of redox balance, inflammatory cascades, metabolism, and other physiological processes.17,22–25 A number of these responses are mediated by the stress-regulated transcription factor CCAAT Homologous Binding Protein (CHOP), the persistent expression of which promotes accumulation of reactive oxygen species and cell death.25 Accordingly, animals lacking CHOP are protected from a wide array of diseases in which ER stress is implicated.26–29

Given the link between ER stress in the SFO and Ang-II-induced hypertension, the importance of AT1R in the SFO in mediating responses to DOCA-salt, and the association of DOCA-salt hypertension with increased brain RAS activity, we hypothesized that the cardinal dipsogenic, pressor and metabolic responses to DOCA-salt might mechanistically involve brain ER stress, and more specifically, ER stress in the SFO.1,2,13 Moreover, CHOP plays a pivotal role in mediating ER stress, and CHOP-deficient mice are protected from diabetes, diabetic nephropathy and atherosclerosis.27,30,31 Consequently, the purpose of this study was to determine if DOCA-salt hypertension is associated with ER stress in the SFO, if blocking ER stress reduces the cardinal phenotypes associated with DOCA-salt hypertension, and if CHOP plays a mechanistic role in mediating physiological responses to DOCA-salt.

Methods

Specific surgical and pharmacological protocols, and detailed methods are provided in the expanded methods section available in the online-only data supplement.

Animals

All studies were approved by the University of Iowa Animal Care and Use Committee and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Standard laboratory chow (Harlan Teklad, NIH-31 modified 6% mouse diet) and tap water were provided ad libitum until the experiments involving deoxycorticosterone (DOCA) were initiated. Mice used in the study included six-week-old wild-type C57BL/6J mice and CHOP-deficient mice that were highly backcrossed (>10 generations) into the C57BL/6J line and maintained by homozygous breeding.32 Consequently, age and sex matched C57BL/6J were used as controls.

Statistics

We analyzed the data with 1- or 2-way ANOVA, with repeated measurements as appropriate. Bonferroni multiple comparisons procedures were used to explore treatment effects. If equal variance or normality failed, we used nonparametric analysis of our data, such as Mann–Whitney U or Wilcoxon tests. We considered differences significant at P<0.05.

Results

Differential MAP, HR and Drinking Responses to ER Stress Reduction

Our initial rationale was to determine whether alleviating ER stress specifically in the brain would alter the changes in mean arterial blood pressure (MAP), heart rate (HR), and saline intake associated with DOCA-salt-induced hypertension. To do this, we administered the chemical chaperone Tauroursodeoxycholic acid (TUDCA) by intracerebroventricular (ICV) infusion, and then examined the changes in MAP and HR induced by DOCA-salt by radiotelemetry. Baseline MAP and HR was the same and displayed normal circadian periodicity in the groups destined for aCSF or TUDCA (Figure 1A). Following three weeks of DOCA-salt, mice treated only with ICV artificial cerebrospinal fluid (aCSF) exhibited a 14±5 mmHg increase in MAP (Figure 1B). TUDCA had no effect on this response (Figure 1B,C). DOCA-salt led to an 80±15 BPM reduction in HR which was significantly attenuated by TUDCA (Figure 1C).

Figure 1. Cardiovascular Responses to ER Stress Blockade.

Figure 1

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MAP and HR were measured by radiotelemetry at baseline before aCSF and TUDCA (A) and after DOCA-salt and either aCSF or TUDCA (B). The change in MAP and HR comparing aCSF to TUDCA is indicated (C). *P<0.05 vs aCSF. All data are mean ± SEM. Group sizes are indicated.

Next, we examined the saline intake response to TUDCA in DOCA-salt and sham mice using the protocol diagrammed in Figure S1. There were no changes in body weight in response to TUDCA or 4-phenylbutyrate (4PBA, Figure S2). Before ICV aCSF or TUDCA, DOCA-salt treated mice exhibited a significant increase in saline intake compared with sham mice (Figure 2A). ICV TUDCA had no effect on saline intake in sham mice. In contrast to its effect on MAP, ICV TUDCA significantly attenuated saline intake in DOCA-salt treated mice. The decrease in saline intake was maintained for as long as the daily ICV injections of TUDCA were maintained, and saline intake increased to pre-TUDCA levels 1-day after TUDCA was discontinued. There was no decrease in saline intake when TUDCA was infused IP (Figure 2B). The decrease in saline intake was not specific to TUDCA, as another chemical chaperone, 4PBA, similarly decreased DOCA-salt-induced saline intake (Figure 2C). The blunted saline intake response to DOCA-salt induced by TUDCA was replicated in a second independent cohort of mice housed in metabolism cages and was accompanied by decreased urine output (Figure S3). These results suggest that DOCA-salt induces ER stress in the brain that contributes to the saline intake and HR responses but not the pressor response caused by DOCA-salt.

Figure 2. Saline Intake Responses to ER Stress Blockade.

Figure 2

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A. Daily saline intake was measured in sham and DOCA-salt-treated mice before and after daily ICV aCSF or TUDCA. *P<0.05 vs aCSF; πP<0.05 vs sham. B. Daily saline intake was measured in DOCA-salt-treated mice before and after daily IP saline or TUDCA. C. Saline intake was measured in DOCA-salt-treated mice before and after ICV aCSF or 4PBA. These data represent the average of 2 days of measurements taken 6–7 days after 4PBA was started. *P<0.05 vs baseline and aCSF. In all experiments, baseline was the average of 3 days of measurements. All measurements were performed in the home cage. Group sizes are indicated. All data are mean ± SEM.

DOCA-salt Causes ER Stress in the SFO and SON

Given the response to chemical chaperones and the importance of the SFO in mediating saline intake responses to DOCA-salt 13,33,34 we next tested whether DOCA-salt induces ER stress in the SFO by measuring CHOP, a key protein increased in response to ER stress. Background immunoreactivity was detected in the SFO of untreated C57BL/6J control mice and CHOP-deficient mice (Figure 3). DOCA-salt caused a marked elevation in CHOP immunoreactivity in the SFO which was similar in magnitude to that caused by tunicamycin and Ang-II (Figure 3). There was virtually no staining for CHOP outside the SFO except for occasional cells in the supraoptic nuclei (SON) and lateral hypothalamus (Figure 3), consistent with the importance of the SON in mediating drinking responses to Ang-II. There was no increase in CHOP detected in the remainder of the hypothalamus, and the thalamus, preoptic area, cortex, and basal ganglia.

Figure 3. DOCA-salt Induces ER Stress in the SFO and SON.

Figure 3

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Representative micrographs of the SFO and SON in control mice (C57 mice, CHOP-deficient mice) and experimental mice (21 days of DOCA-salt, 10 hrs of Tunicamycin [TM], 14 days of Ang-II) showing CHOP protein by immunohistochemistry. Scale bar is 200 μm. These were representative of n=17 for DOCA-salt, C57BL/6, TM, and CHOP-deficient mice, and n=2 for Ang-II.

The presence of ER stress in the SFO was next validated by ultrastructural analysis using electron microscopy. The rough ER appeared normal with extensive parallel membranes in neurons (Figure 4), glia (Figure S4) and ependymal cells (Figure S5) in the SFO of control mice. Large numbers of ribosomes were observed attached to the RER as well as free in the cytoplasm. In contrast, the RER was broken up into short lengths with numerous swellings or vacuole-like structures in all three cells types after DOCA-salt. Very little of the ER had ribosomes attached and there were few free ribosomes in the cytoplasm. In addition, numerous large vacuoles were observed in the cytoplasm in glia and ependymal cells.

Figure 4. Electron Microscopy of the SFO.

Figure 4

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Representative low and high power electron micrographs of a SFO neuron from control and DOCA-salt mice. Arrows indicate rough endoplasmic reticulum. These were representative of n=4 each for DOCA-salt and C57BL/6.

Attenuating ER Stress in the SFO Decreases Saline Intake

To test the role of ER stress in the SFO in mediating the enhanced saline intake response to DOCA-salt, we specifically targeted over-expression of GRP78 (also known as BiP), the most abundant resident ER chaperone, to the SFO using an adenovirus, after first confirming that we can specifically infect the SFO using AdeGFP (Figure S6). AdGRP78 had no effect on body weight at baseline nor in the sham mice (Figure S7). However SFO directed GRP78 lowered body weight by 1.5g in DOCA-salt treated mice. Similar to the effects of chemical chaperone treatment, SFO-specific GRP78 decreased saline intake in DOCA-salt mice but had no effect in sham mice (Figure 5). These data implicate ER stress in the SFO as partially mediating the increase in saline intake induced by DOCA-salt.

Figure 5. Relieving ER Stress in SFO Attenuates DOCA-induced Saline Intake.

Figure 5

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A. Saline intake before (average of 3 days) and after ICV AdGRP78 or AdLacZ (average of days 2–4). *P<0.05 vs AdLacZ. B. Change in saline intake before and after ICV AdGRP78 or AdLacZ. *P<0.05 vs all other groups. Group sizes are indicated. All data are mean ± SEM.

Role of CHOP in Mediating Cardinal Phenotypes of DOCA-salt

Seeing the increase in CHOP immunocytochemistry in the SFO, we next asked if CHOP plays a functional role in mediating responses to DOCA-salt. CHOP-deficiency had no effect on baseline saline intake (Figure 6A), or systolic blood pressure (SBP, Figure 6B) but caused a modest tachycardia (Figure S8). Saline intake was blunted in DOCA-salt-treated CHOP-deficient mice (Figure 6A). SBP was similar comparing DOCA-salt-treated C57 and CHOP-deficient mice (Figure 6B). The bradycardia caused by DOCA-salt was amplified in CHOP-deficient mice (Figure S8). We previously reported that DOCA-salt increases resting metabolic rate (RMR), a finding replicated here (Figure S7).4,5 Interestingly, RMR was elevated under baseline conditions in CHOP-deficient mice which resulted in a blunted response to DOCA-salt (Figure S9).

Figure 6. Role of CHOP in Saline Intake and Blood Pressure after DOCA-salt.

Figure 6

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Average daily saline intake (A) and systolic blood pressure (B) before and after DOCA-salt in C57BL/6 and CHOP-deficient mice. A. **P<0.05 vs C57+DOCA-salt. B. *P<0.05 vs baseline. Group sizes are indicated by the number in each bar. All data are mean ± SEM.

Discussion

The main findings of this study are that DOCA-salt results in 1) ER stress in the SFO and SON, 2) relief of ER stress in the brain by chemical chaperone or in the SFO by adenoviral transduction of a resident molecular chaperone decreases saline intake, but not arterial blood pressure, 3) CHOP may play a mechanistic role in mediating saline intake in response to DOCA-salt, and 4) CHOP may be involved in regulating HR and resting metabolic rate. These data lead us to conclude that DOCA-salt causes ER stress in the brain, specifically in the SFO and SON, which is mechanistically linked to increased saline intake but not hypertension. Thus, the increase in saline intake and arterial pressure by DOCA-salt are mediated by mechanistically distinct intracellular signaling processes that can be separated by the effect induced by ER stress.

The evidence supporting a mechanistic contribution of ER stress to DOCA-salt-induced saline intake is supported by the decrease in saline intake in response to two chemically distinct chaperones injected directly into the brain. Since both agents had their effect when injected ICV, we cannot determine where in brain these agents had their primary effect. It is likely that both agents gained access to regions of the brain outside the blood brain barrier such as the SFO and other circumventricular organs. However, despite being small molecular weight chemicals (both less than 500 daltons) it is unclear if they gain access to the brain parenchyma. That TUDCA injected systemically had no effect on saline intake strongly suggests its effect is mediated by relieving ER stress in the brain. Along these lines, it is interesting to note that similar doses of TUDCA injected intraperitoneal decreased Aβ amyloid deposition in the brain of an Alzheimer’s disease mouse model and reduced microglial activation in a model of acute neuro-inflammation.35,36 Thus the difference in efficacy of intraperitoneal vs intraventricular TUDCA remains difficult to explain. It is also notable that saline intake rapidly returned to normal 1-day after discontinuing TUDCA. Thus, it is possible that a continuous relatively high local concentration of TUDCA may be required to relieve ER stress in the face of continuous DOCA and high salt.

Immunohistochemistry revealed that DOCA-salt elevated the level of CHOP protein in both the SFO and SON, most strongly and consistently in the SFO. The increase in CHOP in the SFO was anticipated for two reasons. First, ER stress in the SFO was previously implicated in mediating the hypertensive response to Ang-II infusion.14 Second, Ang-II AT1 receptors in the SFO are required to mediate fluid and sodium intake responses to DOCA-salt.13 These data are consistent with the well-established role of the SFO in the regulation of salt and water intake.37 Although evidence for ER stress in the SON (as suggested by increase CHOP) was not originally anticipated, it is consistent with its known function to regulate fluid and electrolyte balance through arginine vasopressin.38 Since the SON contains osmosensitive projections from the SFO,39 it will be interesting to assess in future studies if the increase in CHOP immunoreactivity in the SON is restricted to the osmosensitive neurons which project from the SFO. The SFO has a distinctive structure which allows it to sample the chemical constituents of the cerebrospinal fluid, and is uniquely positioned with afferent and efferent connections which allows it to integrate both blood-borne and CNS-derived signals.40 This may make the SFO particularly sensitive to those agents which cause ER stress such as Ang-II, tunicamycin and as shown here DOCA-salt. A recent study employing optogenetics to directly stimulate action potentials in SFO neurons revealed that the increase in water intake occurs virtually immediately after stimulation.41 Consequently, future studies will have to mechanistically determine how DOCA-salt-induced ER stress, which likely develops over time, induces activity of SFO neurons.

It is noteworthy that despite the increase in CHOP immunoreactivity, we were unable to detect a clear difference in CHOP gene expression in the SFO of DOCA-salt treated mice (data not shown). However, it was recently reported that increased expression of the mRNAs encoding several ER stress biomarkers (s-XBP, ATF4, GRP78 [also known as BiP] and CHOP) was evident in the SFO and to a lesser extent the paraventricular nucleus (PVN) of DOCA-salt-treated mice.42 However, unlike their study which surprisingly concluded that ER stress does not occur in the SFO in response to DOCA-salt, our identification of increased CHOP protein in the SFO (and SON) concomitant with ultrastructural changes consistent with ER stress, and decreased saline intake in response to SFO-targeted delivery of AdGRP78 provide compelling evidence for the induction of ER stress in the SFO in response to DOCA-salt.

Perhaps the most interesting finding was that relieving ER stress with TUDCA attenuated DOCA-salt induced saline intake but not hypertension. Whereas, the absence of an effect on blood pressure is consistent with a previous report observing no changes in DOCA-salt induced blood pressure in response to TUDCA 42, it stands in contrast to two other studies where relief of ER stress prevented the hypertensive response in Ang-II 14 and reduced blood pressure in the spontaneously hypertensive rat.43 Assuming there are no technical limitations in both DOCA-salt studies, these results have two main implications. First, ER stress may play a role in the pathogenesis of some forms of hypertension (e.g. Ang-II and some forms of genetic hypertension), but not others (e.g. DOCA-salt hypertension). Chemical chaperones also relieve pulmonary hypertension in some models.44,45 Additional studies will be required to assess the full range of experimental and genetic models of hypertension which involve an ER stress component. Of course, we cannot rule out the possibility that DOCA-salt causes ER stress in a region of the brain which cannot be accessed by ICV TUDCA, perhaps the brainstem. Indeed, the rostral ventrolateral medulla has been implicated as important region mediating DOCA-salt hypertension.46 It is also interesting to speculate that the renin/Ang-II status of the model is important in determining a response to relief of brain ER stress. DOCA-salt is a model of low renin/Ang-II hypertension whereas the Ang-II infusion model and SHR represent models of high and normal Ang-II hypertension, respectively.

The second and perhaps more important implication is that ER stress may be a mechanism which distinguishes some of the common phenotypes observed concurrently in hypertension. In the case of the DOCA-salt model, we refer to the increased saline intake which pre-dates and then accompanies hypertension. Because blood pressure was not reduced concomitantly with saline intake by TUDCA, it might be tempting to speculate that increased saline intake is not necessary for the development or maintenance of DOCA-salt hypertension. However, it is well established that both DOCA and high salt are required to mediate hypertension in this model.47 Blood pressure and saline intake studies were performed in independent cohorts of mice; and one potential limitation of our study was that we used different protocols to study the blood pressure and saline intake responses to TUDCA, ICV mini-pump in the former and daily ICV injections in the latter.

It is quite interesting that the reduction in saline intake, but not blood pressure, induced by TUDCA-mediated relief of ER stress, was paralleled in studies examining mice deficient in CHOP. CHOP-deficient mice exhibited a modest but significant decrease in DOCA-salt-induced saline intake, but no change in the hypertensive response to DOCA-salt. Thus whereas the blood pressure response to DOCA-salt is preserved in CHOP-deficient mice, the saline-intake response is impaired. This suggests the possibility that the full extent of saline-intake induced by DOCA-salt requires ER stress mediated partly through CHOP, and that CHOP is dispensable for DOCA-salt induced hypertension. CHOP is a transcription factor which, with other transcription factors, participates in the transcriptional response to ER stress by increasing and decreasing the expression of other target genes. CHOP expression is induced by the PERK/eiF2/ATF4 and ATF6 branches of the unfolded protein response (UPR). CHOP and XBP1 are concomitantly induced and both were reportedly induced in the SFO in response to DOCA-salt.42 It is well established that prolonged activation of CHOP can induce apoptosis (reviewed in 25,48). We have no evidence addressing if apoptosis is occurring in the brain of DOCA-salt mice and if this is worsened in regions showing evidence of ER stress, such as the SFO and SON. It is also unclear if DOCA-salt treated CHOP-deficient mice are protected from ER-stress induced apoptosis. There is precedence for protection as CHOP-deficient mice were reported to be protected from neuronal apoptosis induced by ischemia, and primary hippocampal neurons from these mice were protected from hypoxia-reoxygenation-induced apoptosis.49 Consequently studies directly examining if apoptosis or apoptosis signaling cascades are activated/repressed would be warranted.

Perspectives

ER stress in the brain has become a new mechanistic paradigm associated with Ang-II-dependent hypertension and has been reported in at least one model of genetic hypertension.14,43 Our studies suggest that ER stress also occurs in the DOCA-salt model of hypertension but that relief of ER stress by chemical chaperones does not lower blood pressure as it does in other models. However, chemical chaperones strongly blunted the saline intake which occurs concomitantly in DOCA-salt hypertension. Although our study focused solely on the brain, ER stress has been reported to occur in endothelial dysfunction, atherosclerosis, in several renal disorders, and in the placenta in preeclampsia.19,50,51 Thus it is likely that like oxidative stress, ER stress plays an important role in the pathogenesis of at least some forms of hypertension. Nevertheless, it remains unclear if ER stress plays an important mechanistic role in human essential hypertension and if either chemical or molecular chaperones will become effective treatments for hypertension.

Supplementary Material

Data Supplement

Novelty and Significance.

What is New?

  • DOCA-salt hypertension is associated with ER stress in the SFO and SON as evidenced by increased CHOP immunoreactivity in both nuclei and altered ultrastructure of the ER in the SFO.

  • Relief of ER stress by brain-specific administration of chemical chaperones decreases DOCA-salt induced saline intake but not hypertension.

  • Relief of ER stress in the SFO be targeted delivery of AdGRP78 decreases DOCA-salt induced saline intake.

  • CHOP-deficient mice exhibit blunted DOCA-salt-induced saline intake.

What is Relevant?

  • ER stress is an essential component of DOCA-salt-induced saline intake.

  • CHOP and it’s transcriptional pathway may play a mechanistic role in mediating increased drinking in the DOCA-salt model of hypertension.

Summary

We conclude that 1) brain ER stress mediates the saline intake, but not blood pressure response to DOCA-salt, 2) DOCA-salt causes ER stress in the SFO which when attenuated by GRP78 blunts saline intake, and 3) CHOP may play a modest functional role in DOCA-salt-induced saline intake. The results suggest a mechanistic distinction between the importance of ER stress in mediating effects of DOCA-salt on saline intake and blood pressure.

Acknowledgments

We thank the University of Iowa Genome Editing Facility for genotyping CHOP-deficient mice, University of Iowa Vector Core for adenoviruses, and to Dr. Colin Young for technical advice. We thank D. Ron (MRC, Cambridge) for CHOP−/− mice and D. McCabe for assistance with CHOP IHC.

Source of funding

This work was supported by the National Institutes of Health (NIH) Program Project Grant HL084207 (CDS, JLG, RLD), HL058048, and HL061446 (CDS), HL063887 to RLD, an NIH Pathway to Independence K99/R00 award HL098276 (JLG), by an American Heart Association Midwest Affiliate Postdoctoral Fellowship 11POST5610024 (AMH), and by the Roy J. Carver Trust (CDS). RLD is the Andrew Dickson White Professor of Molecular Physiology at Cornell University.

Footnotes

Conflict of interest

None

References

  • 1.Itaya Y, Suzuki H, Matsukawa S, Kondo K, Saruta T. Central renin-angiotensin system and the pathogenesis of DOCA-salt hypertension in rats. Am J Physiol. 1986;251:H261–H268. doi: 10.1152/ajpheart.1986.251.2.H261. [DOI] [PubMed] [Google Scholar]
  • 2.Kubo T, Yamaguchi H, Tsujimura M, Hagiwara Y, Fukumori R. Blockade of angiotensin receptors in the anterior hypothalamic preoptic area lowers blood pressure in DOCA-salt hypertensive rats. Hypertens Res. 2000;23:109–118. doi: 10.1291/hypres.23.109. [DOI] [PubMed] [Google Scholar]
  • 3.Park CG, Leenen FH. Effects of centrally administered losartan on deoxycorticosterone-salt hypertension rats. J Korean Med Sci. 2001;16:553–557. doi: 10.3346/jkms.2001.16.5.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Grobe JL, Grobe CL, Beltz TG, Westphal SG, Morgan DA, Xu D, de Lange WJ, Li H, Sakai K, Thedans DR, Cassis LA, Rahmouni K, Mark AL, Johnson AK, Sigmund CD. The Brain Renin-Angiotensin System Controls Divergent Efferent Mechanisms to Regulate Fluid and Energy Balance. Cell Metabolism. 2010;12:431–442. doi: 10.1016/j.cmet.2010.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Grobe JL, Buehrer BA, Hilzendeger AM, Liu X, Davis DR, Xu D, Sigmund CD. Angiotensinergic signaling in the brain mediates metabolic effects of deoxycorticosterone (DOCA)-salt in C57 mice. Hypertension. 2011;57:600–607. doi: 10.1161/HYPERTENSIONAHA.110.165829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Smith PM, Chambers AP, Price CJ, Ho W, Hopf C, Sharkey KA, Ferguson AV. The subfornical organ: a central nervous system site for actions of circulating leptin. Am J Physiol Regul Integr Comp Physiol. 2009;296:R512–R520. doi: 10.1152/ajpregu.90858.2008. [DOI] [PubMed] [Google Scholar]
  • 7.Smith PM, Ferguson AV. Circulating signals as critical regulators of autonomic state--central roles for the subfornical organ. Am J Physiol Regul Integr Comp Physiol. 2010;299:R405–R415. doi: 10.1152/ajpregu.00103.2010. [DOI] [PubMed] [Google Scholar]
  • 8.Cottrell GT, Ferguson AV. Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul Pept. 2004;117:11–23. doi: 10.1016/j.regpep.2003.09.004. [DOI] [PubMed] [Google Scholar]
  • 9.Gonzalez AD, Wang G, Waters EM, Gonzales KL, Speth RC, Van Kempen TA, Marques-Lopes J, Young CN, Butler SD, Davisson RL, Iadecola C, Pickel VM, Pierce JP, Milner TA. Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice. Neuroscience. 2012;226:489–509. doi: 10.1016/j.neuroscience.2012.08.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yang G, Gray TS, Sigmund CD, Cassell MD. The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system. Brain Res. 1999;817:123–131. doi: 10.1016/s0006-8993(98)01236-0. [DOI] [PubMed] [Google Scholar]
  • 11.Sakai K, Agassandian K, Morimoto S, Sinnayah P, Cassell MD, Davisson RL, Sigmund CD. Local production of angiotensin II in the subfornical organ causes elevated drinking. J Clin Invest. 2007;117:1088–1095. doi: 10.1172/JCI31242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sinnayah P, Lazartigues E, Sakai K, Sharma RV, Sigmund CD, Davisson RL. Genetic Ablation of Angiotensinogen in the Subfornical Organ of the Brain Prevents the Central Angiotensinergic Pressor Response. Circ Res. 2006;99:1125–1131. doi: 10.1161/01.RES.0000250259.66683.f5. [DOI] [PubMed] [Google Scholar]
  • 13.Hilzendeger AM, Cassell MD, Davis DR, Stauss HM, Mark AL, Grobe JL, Sigmund CD. Angiotensin type 1a receptors in the subfornical organ are required for deoxycorticosterone acetate-salt hypertension. Hypertension. 2013;61:716–722. doi: 10.1161/HYPERTENSIONAHA.111.00356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Young CN, Cao X, Guruju MR, Pierce JP, Morgan DA, Wang G, Iadecola C, Mark AL, Davisson RL. ER stress in the brain subfornical organ mediates angiotensin-dependent hypertension. J Clin Invest. 2012;122:3960–3964. doi: 10.1172/JCI64583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Schroder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem. 2005;74:739–789. doi: 10.1146/annurev.biochem.73.011303.074134. [DOI] [PubMed] [Google Scholar]
  • 16.Wu J, Kaufman RJ. From acute ER stress to physiological roles of the Unfolded Protein Response. Cell Death Differ. 2006;13:374–384. doi: 10.1038/sj.cdd.4401840. [DOI] [PubMed] [Google Scholar]
  • 17.Rutkowski DT, Hegde RS. Regulation of basal cellular physiology by the homeostatic unfolded protein response. J Cell Biol. 2010;189:783–794. doi: 10.1083/jcb.201003138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wang S, Kaufman RJ. The impact of the unfolded protein response on human disease. J Cell Biol. 2012;197:857–867. doi: 10.1083/jcb.201110131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zhou AX, Tabas I. The UPR in atherosclerosis. Semin Immunopathol. 2013;35:321–332. doi: 10.1007/s00281-013-0372-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Clarke HJ, Chambers JE, Liniker E, Marciniak SJ. Endoplasmic reticulum stress in malignancy. Cancer Cell. 2014;25:563–573. doi: 10.1016/j.ccr.2014.03.015. [DOI] [PubMed] [Google Scholar]
  • 21.Su J, Zhou L, Kong X, Yang X, Xiang X, Zhang Y, Li X, Sun L. Endoplasmic reticulum is at the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in the pathogenesis of diabetes mellitus. J Diabetes Res. 2013;2013:193461. doi: 10.1155/2013/193461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell. 2010;140:900–917. doi: 10.1016/j.cell.2010.02.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal. 2007;9:2277–2293. doi: 10.1089/ars.2007.1782. [DOI] [PubMed] [Google Scholar]
  • 24.Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081–1086. doi: 10.1126/science.1209038. [DOI] [PubMed] [Google Scholar]
  • 25.Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011;13:184–190. doi: 10.1038/ncb0311-184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Thorp E, Li G, Seimon TA, Kuriakose G, Ron D, Tabas I. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe−/− and Ldlr−/− mice lacking CHOP. Cell Metab. 2009;9:474–481. doi: 10.1016/j.cmet.2009.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Song B, Scheuner D, Ron D, Pennathur S, Kaufman RJ. Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes. J Clin Invest. 2008;118:3378–3389. doi: 10.1172/JCI34587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Oyadomari S, Koizumi A, Takeda K, Gotoh T, Akira S, Araki E, Mori M. Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. J Clin Invest. 2002;109:525–532. doi: 10.1172/JCI14550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Gao J, Ishigaki Y, Yamada T, Kondo K, Yamaguchi S, Imai J, Uno K, Hasegawa Y, Sawada S, Ishihara H, Oyadomari S, Mori M, Oka Y, Katagiri H. Involvement of endoplasmic stress protein C/EBP homologous protein in arteriosclerosis acceleration with augmented biological stress responses. Circulation. 2011;124:830–839. doi: 10.1161/CIRCULATIONAHA.110.014050. [DOI] [PubMed] [Google Scholar]
  • 30.Tsukano H, Gotoh T, Endo M, Miyata K, Tazume H, Kadomatsu T, Yano M, Iwawaki T, Kohno K, Araki K, Mizuta H, Oike Y. The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2010;30:1925–1932. doi: 10.1161/ATVBAHA.110.206094. [DOI] [PubMed] [Google Scholar]
  • 31.Wu J, Zhang R, Torreggiani M, Ting A, Xiong H, Striker GE, Vlassara H, Zheng F. Induction of diabetes in aged C57B6 mice results in severe nephropathy: an association with oxidative stress, endoplasmic reticulum stress, and inflammation. Am J Pathol. 2010;176:2163–2176. doi: 10.2353/ajpath.2010.090386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT, Remotti H, Stevens JL, Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev. 1998;12:982–995. doi: 10.1101/gad.12.7.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Coble JP, Johnson RF, Cassell MD, Johnson AK, Grobe JL, Sigmund CD. Activity of PKC-alpha within the Subfornical Organ is Necessary for Fluid Intake due to Brain Angiotensin. Hypertension. 2014;64:141–148. doi: 10.1161/HYPERTENSIONAHA.114.03461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Coble JP, Cassell MD, Davis DR, Grobe JL, Sigmund CD. Activation of the Renin-Angiotensin System Specifically in the Subfornical Organ is Sufficient to Induce Fluid Intake. Am J Physiol Regul Integr Comp Physiol. 2014;307:R376–R386. doi: 10.1152/ajpregu.00216.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Dionisio PA, Amaral JD, Ribeiro MF, Lo AC, D’Hooge R, Rodrigues CM. Amyloid-beta pathology is attenuated by tauroursodeoxycholic acid treatment in APP/PS1 mice after disease onset. Neurobiol Aging. 2015;36:228–240. doi: 10.1016/j.neurobiolaging.2014.08.034. [DOI] [PubMed] [Google Scholar]
  • 36.Yanguas-Casas N, Barreda-Manso MA, Nieto-Sampedro M, Romero-Ramirez L. Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation. J Neuroinflammation. 2014;11:50. doi: 10.1186/1742-2094-11-50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Simpson JB, Routtenberg A. Subfornical organ: site of drinking elicitation by angiotensin II. Science. 1973;181:1772–1775. doi: 10.1126/science.181.4105.1172. [DOI] [PubMed] [Google Scholar]
  • 38.Grindstaff RR, Cunningham JT. Cardiovascular regulation of vasopressin neurons in the supraoptic nucleus. Exp Neurol. 2001;171:219–226. doi: 10.1006/exnr.2001.7745. [DOI] [PubMed] [Google Scholar]
  • 39.Weiss ML, Hatton GI. Collateral input to the paraventricular and supraoptic nuclei in rat. II. Afferents from the ventral lateral medulla and nucleus tractus solitarius. Brain Res Bull. 1990;25:561–567. doi: 10.1016/0361-9230(90)90112-d. [DOI] [PubMed] [Google Scholar]
  • 40.Coble JP, Grobe JL, Johnson AK, Sigmund CD. Mechanisms of brain renin angiotensin system-induced drinking and blood pressure: importance of the subfornical organ. Am J Physiol Regul Integr Comp Physiol. 2015;308:R238–R249. doi: 10.1152/ajpregu.00486.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Oka Y, Ye M, Zuker CS. Thirst driving and suppressing signals encoded by distinct neural populations in the brain. Nature. 2015 doi: 10.1038/nature14108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Xia H, Moreira de QT, Sriramula S, Feng Y, Johnson T, Mungrue IN, Lazartigues E. Brain ACE2 overexpression reduces DOCA-salt hypertension independently of endoplasmic reticulum stress. Am J Physiol Regul Integr Comp Physiol. 2015;308:R370–R380. doi: 10.1152/ajpregu.00366.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Chao YM, Lai MD, Chan JY. Redox-sensitive endoplasmic reticulum stress and autophagy at rostral ventrolateral medulla contribute to hypertension in spontaneously hypertensive rats. Hypertension. 2013;61:1270–1280. doi: 10.1161/HYPERTENSIONAHA.111.00469. [DOI] [PubMed] [Google Scholar]
  • 44.Koyama M, Furuhashi M, Ishimura S, Mita T, Fuseya T, Okazaki Y, Yoshida H, Tsuchihashi K, Miura T. Reduction of endoplasmic reticulum stress by 4-phenylbutyric acid prevents the development of hypoxia-induced pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol. 2014;306:H1314–H1323. doi: 10.1152/ajpheart.00869.2013. [DOI] [PubMed] [Google Scholar]
  • 45.Dromparis P, Paulin R, Stenson TH, Haromy A, Sutendra G, Michelakis ED. Attenuating endoplasmic reticulum stress as a novel therapeutic strategy in pulmonary hypertension. Circulation. 2013;127:115–125. doi: 10.1161/CIRCULATIONAHA.112.133413. [DOI] [PubMed] [Google Scholar]
  • 46.Kubo T, Fukumori R, Kobayashi M, Yamaguchi H. Altered cholinergic mechanisms and blood pressure regulation in the rostral ventrolateral medulla of DOCA-salt hypertensive rats. Brain Res Bull. 1998;45:327–332. doi: 10.1016/s0361-9230(97)00380-8. [DOI] [PubMed] [Google Scholar]
  • 47.Goodwin FJ, Knowlton AI, Laragh JH. Absence of renin suppression by deoxycorticosterone acetate in rats. Am J Physiol. 1969;216:1476–1480. doi: 10.1152/ajplegacy.1969.216.6.1476. [DOI] [PubMed] [Google Scholar]
  • 48.Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006;7:880–885. doi: 10.1038/sj.embor.7400779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, Ushio Y, Mori M. Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 2004;11:403–415. doi: 10.1038/sj.cdd.4401365. [DOI] [PubMed] [Google Scholar]
  • 50.Cindrova-Davies T. The therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins for treatment of preeclampsia. Front Pharmacol. 2014;5:119. doi: 10.3389/fphar.2014.00119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Cunard R, Sharma K. The endoplasmic reticulum stress response and diabetic kidney disease. Am J Physiol Renal Physiol. 2011;300:F1054–F1061. doi: 10.1152/ajprenal.00021.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]

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