Modulation of early gene expression responses to water deprivation stress by the E3 ubiquitin ligase ATL80: implications for retrograde signaling interplay.

BACKGROUND: Primary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of ATL80, an E3 ubiquitin ligase, in the dynamics of gene expression following water deprivation stress. We observed that ATL80 is rapidly activated within minutes of water deprivation stress perception, reaching peak expression around 60 min before gradually declining. ATL80, despite its post-translational regulation role, emerged as a key player in modulating early gene expression responses to water deprivation stress. RESULTS: The impact of ATL80 on gene expression was assessed using a time-course microarray analysis (0, 15, 30, 60, and 120 min), revealing a burst of differentially expressed genes, many of which were associated with various stress responses. In addition, the diversity of early modulation of gene expression in response to water deprivation stress was significantly abolished in the atl80 mutant compared to wild-type plants. A subset of 73 genes that exhibited a similar expression pattern to ATL80 was identified. Among them, several are linked to stress responses, including ERF/AP2 and WRKY transcription factors, calcium signaling genes, MAP kinases, and signaling peptides. Promoter analysis predicts enrichment of binding sites for CAMTA1 and CAMTA5, which are known regulators of rapid stress responses. Furthermore, we have identified a group of differentially expressed ERF/AP2 transcription factors, proteins associated with folding and refolding, as well as pinpointed core module genes which are known to play roles in retrograde signaling pathways that cross-referenced with the early ATL80 transcriptome. CONCLUSIONS: Based on these findings, we propose that ATL80 may target one or more components within the retrograde signaling pathways for degradation. In essence, ATL80 serves as a bridge connecting these signaling pathways and effectively functions as an alarm signal.

Applicability of thermography as a potential non-invasive technique to assess the body-thermal status of heat-stressed and water-deprived goats (Capra hircus).

To evaluate the use of infrared thermography to assess the thermal status of heat-stressed and water-deprived Capra hircus, full-body surface temperature (TS) and six other body-thermal variables [core, rectal (TR), and skin (TSK) temperatures, respiratory and heart rates, and total body-thermal gradient (core-to-ambient, BTG)] were measured after three days of euhydration (EU), dehydration (DE), and rehydration (RE). Results revealed that the combined effect of heat stress and water deprivation had affected all tested variables including the TS, and once these animals gained access to water in the RE stage variables returned to their EU levels. Moreover, there were positive correlations between TS and all variables with the exception of BTG. From these six variables, only three variables (i.e. the TR, TSK, and BTG) during the DE stage and two variables (i.e. the TSK and BTG) throughout the experimental stages showed higher constancy (R2 ≥ 0 75, P < 0 001; agreement intervals ±1 96 95 % CI) with TS. However, BTG appeared more closely correlated with TS, representing the body-thermal status more realistically than other variables. In effect, the mean and thresholds of the BTG were predicted using the recorded TS and were within 0.02 °C of original estimates. Collectively, these findings show that infrared thermography is appropriate for assessing body-thermal status, and thus the welfare, of these animals under the three conditions studied, and conclude that full-body TS can be a surrogate proxy for BTG in these animals. Further experiments are needed to adequately examine the reproducibility of these results under biometeorologically-simulated environments and natural habitats.

Reliability of sucrose preference testing following short or no food and water deprivation-a Systematic Review and Meta-Analysis of rat models of chronic unpredictable stress.

The sucrose preference test is a popular test for anhedonia in the chronic unpredictable stress model of depression. Yet, the test does not always produce consistent results. Long food and water deprivation before the test, while often implemented, confounds the results by introducing unwanted drives in the form of hunger and thirst. We assessed the reliability of the test when only short or no fasting was used. We searched PubMed, Embase, and Web of Science for studies in rats exposed to chronic unpredictable stress that used no more than 6 h of food and/or water deprivation before the test. Sweet consumptions, for stressed and control/antidepressant-treated animals, in 132 studies were pooled using random effects models. We found a decrease in sweet consumption in stressed rats, compared to controls, that was halved when a non-caloric sweetener was used and significantly reduced when sucrose consumption was corrected for body weight. What is more, the length of food and water deprivation was found to confound the effect. The effect was reversed when the stressed rats were treated with antidepressants. Methodological strategies meant to control for recognized sources of bias when conducting the test were often missing, and so was a clear and complete report of essential study information. Our results indicate that not only is food and water deprivation before the test unnecessary, but not recommended. Even in absence of long fasting, we found evidence of an additional effect on sweet consumption that is unrelated to anhedonia. Without properly controlling for non-hedonic drivers of consumption, the test is unreliable as a proxy measure of anhedonia. Strengthening the methodological rigor and addressing the confounding effect of metabolic factors in the sucrose preference test prevents misleading conclusions that harm the translatability of the associated research and perpetuates the use of animals for little gain.

Water deprivation-induced hypoxia and oxidative stress physiology responses in respiratory organs of the Indian stinging fish in near coastal zones.

Background: Water deprivation-induced hypoxia stress (WDIHS) has been extensively investigated in numerous fish species due to their adaptation with accessory respiratory organs to respire air but this has not been studied in Indian stinging fish Heteropneustes fossilis. Data regarding WDIHS-induced metabolism in accessory respiratory organ (ARO) and gills and its relationship with oxidative stress (OS) in respiratory organs of air-breathing fish H. fossilis, are limited. So, this study aimed to investigate the effects of WDIHS (0, 3, 6, 12, and 18 h) on hydrogen peroxide (H2O2) as reactive oxygen species (ROS), OS, redox regulatory enzymes, and electron transport enzymes (ETC) in ARO and gills of H. fossilis. Methods: Fish were exposed to air for different hours (up to 18 h) against an appropriate control, and ARO and gills were sampled. The levels of oxygen saturation in the body of the fish were assessed at various intervals during exposure to air. Protein carbonylation (PC) and thiobarbituric acid reactive substances (TBARS) were used as OS markers, H2O2 as ROS marker, and various enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), along with the assessment of complex enzymes (I, II, III, and V) as well as the levels of ascorbic acid (AA) and the reduced glutathione (GSH) were quantified in both the tissues. Results: Discriminant function analyses indicate a clear separation of the variables as a function of the studied parameters. The gills exhibited higher levels of GSH and H2O2 compared to ARO, while ARO showed elevated levels of PC, TBARS, AA, SOD, CAT, and GPx activities compared to the gills. The activities of GR and ETC enzymes exhibited similar levels in both the respiratory organs, namely the gills, and ARO. These organs experienced OS due to increased H2O2, TBARS, and PC levels, as observed during WDIHS. Under WDIHS conditions, the activity/level of CAT, GPx, GR, and GSH decreased in ARO, while SOD activity, along with GR, GSH, and AA levels decreased in gills. However, the activity/level of SOD and AA in ARO and CAT in gills was elevated under WDIHS. Complex II exhibited a positive correlation with WDIHS, while the other ETC enzymes (complex I, III, and V) activities had negative correlations with the WDIHS. Discussion: The finding suggests that ARO is more susceptible to OS than gills under WDIHS. Despite both organs employ distinct redox regulatory systems to counteract this stress, their effectiveness is hampered by the inadequacy of small redox regulatory molecules and the compromised activity of the ETC, impeding their ability to effectively alleviate the stress induced by the water-deprivation condition.

Prey consumption does not restore hydration state but mitigates the energetic costs of water deprivation in an insectivorous lizard.

To cope with limited availability of drinking water in their environment, terrestrial animals have developed numerous behavioral and physiological strategies including maintaining an optimal hydration state through dietary water intake. Recent studies performed in snakes, which are generalist carnivorous reptiles, suggest that the benefits of dietary water intake are negated by hydric costs of digestion. Most lizards are generalist insectivores that can shift their prey types, but firm experimental demonstration of dietary water intake is currently missing in these organisms. Here, we performed an experimental study in the common lizard Zootoca vivipara, a keystone mesopredator from temperate climates exhibiting a great diversity of prey in its mesic habitats, in order to investigate the effects of food consumption and prey type on physiological responses to water deprivation. Our results indicate that common lizards cannot improve their hydration state through prey consumption, irrespective of prey type, suggesting that they are primarily dependent upon drinking water. Yet, high-quality prey consumption reduced the energetic costs of water deprivation, potentially helping lizards to conserve a better body condition during periods of limited water availability. These findings have important implications for understanding the physiological responses of ectotherms to water stress, and highlight the complex interactions between hydration status, energy metabolism and feeding behavior in insectivorous lizards.

Use of copeptin in interpretation of the water deprivation test.

INTRODUCTION: Currently, the water deprivation test remains the standard method for distinguishing primary polydipsia (PP) from cranial diabetes insipidus (cDI) and nephrogenic diabetes insipidus (nDI). There is increasing interest in a direct estimate of antidiuretic hormone using plasma copeptin as a stable and reliable surrogate marker. We present our experience of measuring copeptin during the water deprivation test. METHODS: Forty-seven people (17 men) underwent a standard water deprivation test between 2013 and 2021. Plasma copeptin was measured at the start of the test and at the end of the period of water deprivation (maximum osmotic stimulation). Results were classified using prespecified diagnostic criteria. As it is known that a significant proportion of tests will reveal indeterminate results, a final diagnosis was obtained by including relevant pre- and post-test clinical criteria. This diagnosis was then used to plan individual treatment. RESULTS: Basal and stimulated copeptin were significantly higher in the nephrogenic DI group than other categories (p < .001). There was no significant difference in basal or stimulated copeptin between PP, cDI or partial DI. Nine results were indeterminate where the serum and urine osmolality did not give a unified diagnosis. Stimulated copeptin was helpful in reclassifying these patients into the final diagnostic groups. CONCLUSION: Plasma copeptin has additional clinical utility in interpretation of the water deprivation test and may continue to have a place alongside newer stimulation tests.

Water deprivation induces hypoactivity in rats independently of oxytocin receptor signaling at the central amygdala.

Introduction: Vasopressin (AVP) and oxytocin (OXT) are neuropeptides produced by magnocellular neurons (MCNs) of the hypothalamus and secreted through neurohypophysis to defend mammals against dehydration. It was recently demonstrated that MCNs also project to limbic structures, modulating several behavioral responses. Methods and Results: We found that 24 h of water deprivation (WD) or salt loading (SL) did not change exploration or anxiety-like behaviors in the elevated plus maze (EPM) test. However, rats deprived of water for 48 h showed reduced exploration of open field and the closed arms of EPM, indicating hypoactivity during night time. We evaluated mRNA expression of glutamate decarboxylase 1 (Gad1), vesicular glutamate transporter 2 (Slc17a6), AVP (Avpr1a) and OXT (Oxtr) receptors in the lateral habenula (LHb), basolateral (BLA) and central (CeA) amygdala after 48 h of WD or SL. WD, but not SL, increased Oxtr mRNA expression in the CeA. Bilateral pharmacological inhibition of OXTR function in the CeA with the OXTR antagonist L-371,257 was performed to evaluate its possible role in regulating the EPM exploration or water intake induced by WD. The blockade of OXTR in the CeA did not reverse the hypoactivity response in the EPM, nor did it change water intake induced in 48-h water-deprived rats. Discussion: We found that WD modulates exploratory activity in rats, but this response is not mediated by oxytocin receptor signaling to the CeA, despite the upregulated Oxtr mRNA expression in that structure after WD for 48 h.

Motivational state-dependent renewal and reinstatement of operant responding under food and water deprivation states.

Most studies of operant relapse phenomena have studied the role of exteroceptive discriminative features of context in the recovery of behavior after extinction. Interoceptive stimuli (i.e., stimuli arising from the bodies of organisms) may also serve as a part of learning contexts and contribute to relapse. Recent studies have demonstrated that events that function as motivating operations (MOs) may contribute to relapse both by a) eliciting interoceptive conditions that serve as a discriminative context for relapse (i.e., discriminative function) and b) altering the amount of operant responding in the presence of discriminative stimuli associated with reinforcers (i.e., motivational function). The current study examined interactions between these functions of food and water deprivation MOs in the relapse of operant behavior. During acquisition of an operant response, one group of mice was food-deprived and another was water-deprived. The groups then received extinction sessions under the opposite condition. Renewal and reinstatement tests were conducted under both conditions, and more renewal and reinstatement were observed in the motivational states in which acquisition occurred for each. These results are discussed in the context of state-dependent learning.

Differences of nitrogen metabolism in date palm (Phoenix dactylifera) seedlings subjected to water deprivation and salt exposure.

Drought and salt exposure are among the most prevalent and severe abiotic stressors causing serious agricultural yield losses, alone and in combination. Little is known about differences and similarities in the effects of these two stress factors on plant metabolic regulation, particularly on nitrogen metabolism. Here, we studied the effects of water deprivation and salt exposure on water relations and nitrogen metabolites in leaves and roots of date palm seedlings. Both, water deprivation and salt exposure had no significant effects on plant water content or stable carbon (C) and nitrogen (N) isotope signatures. Significant effects of water deprivation on total C and N concentrations were only observed in roots, i.e., decreased total C and increased total N concentrations. Whereas salt exposure initially decreased total C and increased total N concentrations significantly in roots, foliar total C concentration was increased upon prolonged exposure. Initially C/N ratios declined in roots of plants from both treatments and upon prolonged salt exposure also in the leaves. Neither treatment affected soluble protein and structural N concentrations in leaves or roots, but resulted in the accumulation of most amino acids, except for glutamate and tryptophan, which remained stable, and serine, which decreased, in roots. Accumulation of the most abundant amino acids, lysine and proline, was observed in roots under both treatments, but in leaves only upon salt exposure. This finding indicates a similar role of these amino acids as compatible solutes in the roots in response to salt und drought, but not in the leaves. Upon prolonged treatment, amino acid concentrations returned to levels found in unstressed plants in leaves of water deprived, but not salt exposed, plants. The present results show both water deprivation and salt exposure strongly impact N metabolism of date palm seedlings, but in a different manner in leaves and roots.

Alterations in Kidney Structures Caused by Age Vary According to Sex and Dehydration Condition.

Aging is a complex biological process, resulting in gradual and progressive decline in structure and function in many organ systems. Our objective is to determine if structural changes produced by aging vary with sex in a stressful situation such as dehydration. The expression of Slc12a3 mRNA in the renal cortex, α-smooth muscle actin (α-SMA), and fibronectin was evaluated in male and female rats, aged 3 and 18 months, submitted and not submitted to water deprivation (WD) for 48 h, respectively. When comparing ages, 18-month-old males showed a lower expression of Slc12a3 mRNA than 3-month-old males, and control and WD 18-month-old male and female rats exhibited a higher expression of α-SMA than the respective 3-month-old rats. Fibronectin was higher in both control and WD 18-month-old males than the respective 3-month-old males. In females, only the control 18-month-old rats showed higher fibronectin than the control 3-month-old rats. When we compared sex, control and WD 3-month-old female rats had a lower expression of Slc12a3 mRNA than the respective males. The WD 18-month-old male rats presented a higher expression of fibronectin and α-SMA than the WD 18-month-old female rats. When we compared hydric conditions, the WD 18-month-old males displayed a lower relative expression of Slc12a3 mRNA and higher α-SMA expression than the control 18-month-old males. Aging, sex, and dehydration lead to alterations in kidney structure.

[Diagnosis and tactics of managing a patient with central diabetes insipidus on the example of a clinical case].

Diagnosis and differential diagnosis of patients with diabetes insipidus is often a difficult task for the endocrinologist. This case report focuses on a patient with a long history of central idiopathic diabetes insipidus who had a substantial decrease in desmopressin requirements during the last year of follow-up. Conducting tests with osmotic stimulation (test with water deprivation, infusion test with hypertonic solution) made it possible to answer the question of the persistence of the disease, as well as to determine a further management plan, taking into account the physiological characteristics of our patient.

High-fat diet changes the behavioural and hormonal responses to water deprivation in male Wistar rats.

NEW FINDINGS: What is the central question of this study? What is the effect of an obesogenic diet on the control of hydromineral balance in rats? What is the main finding and its importance? The results showed that, when dehydrated, rats fed a high-fat diet drink less water than their control-diet-fed counterparts. Changes in aquaporin-7 and peroxisome proliferator-activated receptor α expression in the white adipose tissue might be involved. ABSTRACT: High-fat diet (HFD) increases fat accumulation, glycaemia and blood triglycerides and is used as a model to study obesity. Besides the metabolic changes, obesity likely affects water intake. We assessed the effects of HFD on behavioural and hormonal responses to water deprivation. Additionally, we measured if the adipose tissue is differentially affected by water deprivation in control and HFD-fed rats. HFD rats showed a decreased basal water intake when compared to control-fed rats. When subjected to 48 h of water deprivation, as expected, both control and HFD rats drank more water than the hydrated rats. However, the increase in water intake was lessened in HFD dehydrated rats. Similarly, the increase in haematocrit in dehydrated rats was less pronounced in HFD dehydrated rats. These results suggest that HFD diminishes drinking behaviour. White adipose tissue weight, glycaemia and plasma glycerol concentration were increased in HFD rats; however, after 48 h of water deprivation, these parameters were significantly decreased in dehydrated HFD rats, when compared to controls. The increase in adipose tissue caused by HFD may mitigate the effects of dehydration, possibly through the increased production of metabolic water caused by lipolysis in the adipocytes. Oxytocin possibly mediates the lipolytic response, since both its secretion and receptor expression are affected by dehydration in both control and HFD rats, which suggests that oxytocin signalling is maintained in these conditions. Changes in mediators of lipolysis, such as aquaporin-7 and peroxisome proliferator-activated receptor α, might contribute to the different effects observed in control and HFD rats.

Uromodulin Regulates Murine Aquaporin-2 Activity via Thick Ascending Limb-Collecting Duct Cross-Talk during Water Deprivation.

Uromodulin, a urinary protein synthesized and secreted from the thick ascending limb (TAL) of the loop of Henle, is associated with hypertension through the activation of sodium reabsorption in the TAL. Uromodulin is a potential target for hypertension treatment via natriuresis. However, its biological function in epithelial cells of the distal nephron segment, particularly the collecting duct, remains unknown. Herein, we examined the regulation of uromodulin production during water deprivation in vivo as well as the effect of uromodulin on the activity of the water channel aquaporin-2 (AQP2) in vitro and in vivo using transgenic mice. Water deprivation upregulated uromodulin production; immunofluorescence experiments revealed uromodulin adhesion on the apical surface of the collecting duct. Furthermore, the activation of AQP2 was attenuated in mice lacking uromodulin. Uromodulin enhanced the phosphorylation and apical trafficking of AQP2 in mouse collecting duct cells treated with the vasopressin analog dDAVP. The uromodulin-induced apical trafficking of AQP2 was attenuated via endocytosis inhibitor treatment, suggesting that uromodulin activates AQP2 through the suppression of endocytosis. This study provides novel insights into the cross-talk between TAL and the collecting duct, and indicates that the modulation of uromodulin is a promising approach for diuresis and hypertension treatment.

Sucrose intake and preference by Wistar Han rats are not influenced by sex or food/water deprivation.

Anhedonia is the decreased ability to experience pleasure from rewarding or enjoyable activities, a core symptom of depression. The sucrose preference test (SPT), based on a two-bottle choice paradigm, is a widely used behavioural paradigm for the evaluation of anhedonia in rodents. Up to now, different protocols have been reported regarding water/food deprivation and duration of exposure to the water/sucrose solutions. In this work, by comparing six of the most used SPT protocols regarding sucrose preference and total intake, in both male and female Wistar Han rats, we showed (i) food/water deprivation does not significantly impact sucrose intake and preference; (ii) increasing the duration of the test is associated with an increased sucrose preference and (iii) no sex-specific differences in the basal sucrose preference of Wistar Han rats. Our results call for standardization of protocols and suggest a protocol without food/water deprivation and a 12-hour duration (lights out) as more efficacious in the measurement of anhedonia in rodents. This protocol not only reduces the confounding factors of drinking patterns and the stress-inducing food/water deprivation but also is not sensitive to sex-specific differences in the total intake of liquid in Wistar Han rats.

Long-duration transit and food and water deprivation alter behavioral activities and aggressive interactions at the feed bunk in beef feedlot steers.

The objective of these experiments was to assess the effects of food and water deprivation and transit duration on the behavior of beef feedlot steers. In Experiment 1, 36 Angus-cross steers (353 ± 10 kg) were stratified to 6 pens and assigned one of three treatments (n = 12 steers per treatment): control (CON; stayed in home pens with ad libitum access to feed and water), deprived (DEPR; stayed in home pens but deprived of feed and water for 18 h), or transported (TRANS; subjected to 18-h transit event and returned to home pens). In Experiment 2, 60 Angus-cross steers (398 ± 5 kg; 6 steers per pen) were transported either 8 (8H) or 18 (18H) h. Four 8H pens (n = 24 steers) and six 18H pens (n = 36 steers) were used for behavioral analysis. In both experiments, the time to eat, drink, and lay down was recorded for each steer upon return to home pens. Total pen displacements from the feed bunk were also assessed for the 2 h following feed access in both experiments. Data were analyzed using Proc Mixed of SAS 9.4, with treatment as a fixed effect. Steer was the experimental unit for behavioral activities, while pen was the experimental unit for bunk displacements. Displacements were analyzed as repeated measures with the repeated variable of time. In Experiment 1, the time to eat and drink was similar across treatments (P ≥ 0.17). However, TRANS laid down in 16.5 min while DEPR did not lay down until 70.5 min post-arrival to pen (P < 0.01). Deprived steers had greater bunk displacements in the first 70 min post-feed access than CON or TRANS, though displacements among treatments from 100 to 120 min post-feed access were similar (treatment × time: P = 0.02). In Experiment 2, both 8H and 18H steers laid down approximately 25 min post-home pen arrival (P = 0.14). There was no effect of transit duration or duration by time on bunk displacements (P ≥ 0.20), though displacements were greater from 0 to 20 min than from 20 to 30 min post-feed access (time: P = 0.04). Steers that were deprived of feed and water were highly motivated to access those resources, while transported steers prioritized laying down. Producers should consider these priorities when preparing to receive cattle from a long transit event.

Because of the segmentation of the cattle industry, cattle are transported at least once during their lives. The objective of these two studies was to determine if transportation, feed and water deprivation, and/or transit duration changed the behavior of feedlot steers. The first study found steers transported for 18 h preferred to lay down instead of competing for food, unlike steers that were deprived of food and water for 18 h. Bunk displacements were also increased in steers deprived of food and water, indicating increased aggression. In the second study examining effects of transit duration (8 vs. 18 h), steers from both treatments laid down within 25 min of arrival back to the home pens. There were no differences in the frequency of bunk displacements between treatments. Producers should consider the increased motivation for cattle to lay down after transportation and the increased aggression at the feed bunk in food-deprived cattle when developing post-arrival management strategies.

Water Deprivation Induces Biochemical Changes Without Reduction in the Insecticidal Activity of Maize and Soybean Transgenic Plants.

Like conventional crops, transgenic plants expressing insecticidal toxins from Bacillus thuringiensis (Bt) are subjected to water deprivation. However, the effects of water deprivation over the insecticidal activity of Bt plants are not well understood. We submitted Bt maize and Bt soybean to water deprivation and evaluated biochemical stress markers and the insecticidal activity of plants against target insects. Bt maize (DAS-Ø15Ø7-1 × MON-89Ø34-3 × MON-ØØ6Ø3-6 × SYN-IR162-4) containing the PowerCore Ultra traits, Bt soybean (DAS-444Ø6-6 × DAS-81419-2) with the Conkesta E3 traits, and commercial non-Bt cultivars were cultivated and exposed to water deprivation in the greenhouse. Leaves were harvested for quantification of hydrogen peroxide, malondialdeyde (MDA), and total phenolics and insecticidal activity. Maize or soybean leaf disks were used to evaluate the insecticidal activity against, respectively, Spodoptera frugiperda (J.E Smith) and Chrysodeixis includens (Walker) neonates. Except for Bt soybean, water deprivation increased hydrogen peroxide and MDA contents in Bt and non-Bt plants. Both biochemical markers of water deficit were observed in lower concentrations in Bt plants than in non-Bt commercial cultivars. Water deprivation did not result in changes of phenolic contents in Bt and non-Bt maize. For Bt or non-Bt soybean, phenolic contents were similar despite plants being exposed or not to water deprivation. Water deprivation did not alter substantially insect survival in non-Bt maize or non-Bt soybean. Despite water deprivation-induced biochemical changes in plants, both Bt plants maintained their insecticidal activity (100% mortality) against the target species.

Transcription Factor TonEBP Stimulates Hyperosmolality-Dependent Arginine Vasopressin Gene Expression in the Mouse Hypothalamus.

The hypothalamic neuroendocrine system is strongly implicated in body energy homeostasis. In particular, the degree of production and release of arginine vasopressin (AVP) in the hypothalamus is affected by plasma osmolality, and that hypothalamic AVP is responsible for thirst and osmolality-dependent water and metabolic balance. However, the osmolality-responsive intracellular mechanism within AVP cells that regulates AVP synthesis is not clearly understood. Here, we report a role for tonicity-responsive enhancer binding protein (TonEBP), a transcription factor sensitive to cellular tonicity, in regulating osmosensitive hypothalamic AVP gene transcription. Our immunohistochemical work shows that hypothalamic AVP cellular activity, as recognized by c-fos, was enhanced in parallel with an elevation in TonEBP expression within AVP cells following water deprivation. Interestingly, our in vitro investigations found a synchronized pattern of TonEBP and AVP gene expression in response to osmotic stress. Those results indicate a positive correlation between hypothalamic TonEBP and AVP production during dehydration. Promoter and chromatin immunoprecipitation assays confirmed that TonEBP can bind directly to conserved binding motifs in the 5'-flanking promoter regions of the AVP gene. Furthermore, dehydration- and TonEBP-mediated hypothalamic AVP gene activation was reduced in TonEBP haploinsufficiency mice, compared with wild TonEBP homozygote animals. Therefore, our result support the idea that TonEBP is directly necessary, at least in part, for the elevation of AVP transcription in dehydration conditions. Additionally, dehydration-induced reductions in body weight were rescued in TonEBP haploinsufficiency mice. Altogether, our results demonstrate an intracellular machinery within hypothalamic AVP cells that is responsible for dehydration-induced AVP synthesis.

PLCδ1 Plays Central Roles in the Osmotic Activation of ΔN-TRPV1 Channels in Mouse Supraoptic Neurons and in Murine Osmoregulation.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

Simplified and improved fluid deprivation test for diagnosing diabetes insipidus.

OBJECTIVE: The challenge of finding patients with the rare conditon of diabetes insipidus in need of vasopressin treatment is demanding. The guidelines for performing the fluid deprivation test and interpreting the results are abundant. We evaluated the discriminative capacity of the fluid deprivation test in patients with polyuria to define a cut off for a more effective discrimination between diabetes insipidus and other polyuria syndromes. RESEARCH DESIGN AND METHODS: Retrospective review and data collection of all ambulatory fluid deprivation tests, of patients with mild polyuria and polydipsia (< 3 L/day), performed between 2000 and 2018. Serum osmolality, urine osmolality, urine volumes and clinical information of diagnosis were retrieved from the patient's medical records. RESULTS: The study group consisted of 153 patients, 123 were diagnosed with non-diabetes insipidus and 30 with diabetes insipidus. After 12 h fasting (baseline) median duration of the fluid deprivation test was 5 h (fasting range: 12-21 h). At baseline, there was a significant difference between median serum and urine osmolality between the groups (P < 0.05). The best cut-off for the diagnosis of diabetes insipidus, was the combination of < 400 mosmol/kg in urine and > 302 mosmol/kg in serum. With this cut-off a sensitivity of 90% and specificity of 98% was achieved. CONCLUSION: After 12 h fasting our proposed cut off clearly differentiated between diabetes insipidus, and non-diabetes insipidus suggesting a possibility to considerably reduce the duration of the fluid deprivation test.

How predictive is body weight on fluid intake in rats? It depends on sex.

The assumption that body weight is a predictor of fluid intake is often used as rationale for normalizing intake to body weight when examining sex differences in drinking behavior. Nonuniform application of this body weight correction likely contributes to discrepancies in the literature. We, however, previously demonstrated sex differences in the relationship between body weight and angiotensin II (AngII)-stimulated water intake. Only after a pharmacological dose of AngII did water intake correlate with body weight, and only in males. Here we investigated whether body weight correlated with fluid intake stimulated by additional dipsogenic agents in male and female rats. We found that intake stimulated by either water deprivation or furosemide correlated with body weight in male rats. We found no relationship between intake and body weight after water deprivation, furosemide treatment, or isoproterenol treatment in females, nor did we find a relationship between intake and body weight after hypertonic saline treatment in either males or females. Finally, we report that daily water intake correlated with body weight in females. This effect, however, is likely the result of a relationship between body weight and food intake because when food was absent or reduced, the correlation between body weight and intake disappeared. These results demonstrate that multiple factors need to be considered when determining the best way to compare fluid intake between males and females and provides insight to help explain the discrepancies in the literature regarding sex differences in fluid intake.