Sodium appetite and thirst do not require angiotensinogen production in astrocytes or hepatocytes

Abstract

In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue‐specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. imageKey points Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain‐specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double‐deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver‐specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin‐deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.

HSD2 neurons are evolutionarily conserved and required for aldosterone-induced salt appetite

“…Switching rats to a low-sodium diet for one week is a simple and ethologically relevant way to boost aldosterone production, activate HSD2 neurons, and increase sodium appetite (34, 54, 60). These effects are uncharacterized in mice, so we first assessed the impact of sodium deprivation on HSD2 neurons and sodium appetite.…”

“…Also, the angiotensin receptor blocker losartan (20 mg/kg) reduced licking for saline after chemogenetic stimulation of HSD2 neurons in mice (35). However, eliminating angiotensin production in the liver and brain did not reduce sodium appetite in mice (54). Aldosterone infusion potently suppresses renin release and angiotensin II production (92), so the renin-angiotensin system probably plays little to no role in aldosterone-induced sodium appetite.…”

“…We did not vary placement of the water bottle (right) and 3% NaCl bottle (left) because appetite, not gustatory discrimination, was the subject of this study. We used 3% (0.51 M) NaCl, which provides optimal signal-to-noise for assessing sodium appetite; mice and rats have a baseline aversion to this saline concentration and typically drink zero to less than 0.1 mL per day unless sodium appetite is provoked (36, 53, 54). At the start of every experiment, each BioDAQ cage also had fresh ALPHA-dri+ Plus bedding (Shepherd Specialty Papers), an overhead wire hopper with standard rodent chow (Teklad 7013), and two bottles (distilled water and 3% NaCl) with sipper tubes, accessible through vestibules in the cage wall, that can be blocked by a gate.…”

“…We exported continuous fluid intake records from the BioDAQ DataViewer (Research Diets), then used Microsoft Excel to organize the data and to calculate total intake volumes of water and 3% NaCl. After acute chemogenetic stimulation with CNO and after week-long dietary sodium deprivation, we analyzed the first 6 hours of access to 3% NaCl, as in previous work (54). For all multi-day infusion protocols, we analyzed 3% NaCl and water intake in 24-hour bins.…”