Antidiuretic Hormone Vasopressin Research Articles

Desmopressin use in major cardiac surgery is associated with renal impairment: a retrospective single-center analysis

BackgroundDesmopressin acetate (1-deamino-8-d-arginine vasopressin—DDAVP) is a analogue of the antidiuretic hormone vasopressin. DDAVP is suggested to reduce bleeding after cardiac surgery using cardiopulmonary bypass. The aim of this study was to determine if DDAVP has any negative impact on renal function leading to acute kidney injury (AKI) and therefore increases the need for renal replacement therapy (RRT).MethodsWe performed a retrospective single institutional cohort analysis of 2,179 patients undergoing elective and urgent cardiac surgery with CPB from 2017 to 2021. Logistic regression analysis was used to investigate any association between DDAVP, the incidence of AKI KDIGO class 3 and the need for RRT, respectively. The model was adjusted for relevant covariates, including preexisting renal impairment, pharmacological hemodynamic support with vasopressors, complexity of the surgery and postoperative lactate. Secondary outcomes included, in hospital mortality and the need for allogenic blood transfusion.ResultsA total of 992 (45.5%) patients received DDAVP intraoperatively during surgery or shortly thereafter. The use of DDAVP was associated with a significant increase in in AKI KDIGO class 3 (OR 2.27; 95% CI 1.46–3.55; p < 0,001) and the need for RRT (OR 2.19; 95%CI 1.48–3.24; p < 0,001). Both findings persisted after covariate adjusting. No increased in-hospital mortality was associated with DDAVP.ConclusionIn cardiac surgery, the use of DDAVP was associated with an increased rate of server AKI and the requirement for RRT. Given the severity of the potential harm associated with DDAVP, an evidence-based reevaluation is needed to enable an accurate risk and benefit assessment.

Dysregulation of the fluid homeostasis system by aging.

Chronic dehydration is a leading cause of morbidity for the elderly, but how aging alters the fluid homeostasis system is not well understood. Here, we used a combination of physiologic, behavioral and circuit analyses to characterize how fluid balance is affected by aging in mice. We found that old mice have a primary defect in sensing and producing the anti-diuretic hormone vasopressin, which results in chronic dehydration. Recordings and manipulations of the thirst circuitry revealed that old mice retain the ability to sense systemic cues of dehydration but are impaired in detecting presystemic, likely oropharyngeal, cues generated during eating and drinking, resulting in disorganized drinking behavior on short timescales. Surprisingly, old mice had increased drinking and motivation after 24-hour water deprivation, indicating that aging does not result in a general impairment in the thirst circuit. These findings reveal how a homeostatic system undergoes coordinated changes during aging.

Lysine Acetylation of Aquaporin-3 in Renal Principal Cell Mediates Water Homeostasis

The kidneys function to maintain water homeostasis. High plasma osmolality activates the release of the antidiuretic hormone vasopressin (AVP), which acts on the kidneys through its receptor AVPR2 on the principal cell (PC) to increase apical aquaporin-2 (AQP2) and basolateral AQP3 abundance for enhanced water reabsorption. However, unlike AQP2, the regulation mechanism(s) of AQP3 is unclear. We previously identified a novel AQP3 post-translational modification: acetylation of lysine 282 (K282), which we hypothesize to positively regulate PC water permeability. We developed mouse models of acetylated AQP3 by mutating K282 to glutamine (K282Q; Q mutant) and deacetylated AQP3 by mutating K282 to arginine (K282R; R mutant), on C57BL/6J background to determine the functional consequences of lysine acetylated AQP3. Male and female mice (10-15 weeks old) received a standard chow diet and free access to water. We first investigated the responses to endogenous AVPR2 activation by subjecting the mice to 18-hr overnight water deprivation and compared to mice with ad libitum water access (ad lib). All dehydrated mice (males WT 3087 ± 291 mOsm/kg, n = 8; Q 3688 ± 113 mOsm/kg, n = 6; R 3447 ± 171 mOsm/kg, n = 11; females WT 3393 ± 266 mOsm/kg, n = 8; Q 3586 ± 206 mOsm/kg, n = 9; R 3749 ± 126 mOsm/kg, n = 10) had higher urine osmolality than ad lib mice (males WT 2409 ± 165 mOsm/kg, n = 8; Q 2428 ± 316 mOsm/kg, n = 7; R 1960 ± 196 mOsm/kg, n = 7; females WT 2281 ± 96 mOsm/kg, n = 8; Q 1935 ± 190 mOsm/kg, n = 11; R 2279 ± 249, n = 7, two-way ANOVA p[treatment] &lt;0.0001 for both sexes). However, there were no significant differences among the genotypes in both sexes [two-way ANOVA p[sex] &gt; 0.05). For plasma osmolality, all dehydrated mice in both sexes (males WT 332 ± 2 mOsm/kg, n = 10; Q 324 ± 2 mOsm/kg, n = 8; R 323 ± 2 mOsm/kg n = 11; females WT 316 ± 3 mOsm/kg, n =10; Q 320 ± 2 mOsm/kg, n = 10; R 308 ± 3 mOsm/kg, n = 11) had higher values than ad lib mice (males WT 314 ± 3 mOsm/kg, n = 10; Q 309 ± 1 mOsm/kg, n = 9; R 313 ± 3 mOsm/kg n = 10; females WT 312 ± 2 mOsm/kg, n =11; Q 305 ± 2 mOsm/kg, n = 11; R 301 ± 4 mOsm/kg, n = 10, 2-way ANOVA p[treatment] &lt; 0.0001 for males and = 0.0006 for females). Female R mutants had lower plasma osmolality than WTs and Q in both ad lib and dehydrated groups (2-way ANOVA p[genotype] = 0.0045). In males, compared to WTs, Q and R mutants had similar plasma osmolality in the ad lib but lower in dehydrated group (2-way ANOVA p[genotype] = 0.0359). This suggests that deacetylated AQP3 may result in a reset osmostat, although this remains to be explored further. We next compared the responses of WT and Q mutants to 4-hour exogenous AVPR2 activation by dDAVP (10 ng, i.p). Q females exhibited lower plasma osmolality than WT females (286 ± 4 vs. 305 ± 2 mOsm/kg, n = 11 for both, Student’s t-test p = 0.0005) suggesting lysine acetylation of AQP3 leads to acute enhancement of water permeability. However, there were no significant differences between Q and WT males (300 ± 5 (n = 7) vs. 303 ± 2 mOsm/kg (n = 8), Student’s t-test p &gt; 0.05). Therefore, we conclude that lysine acetylation of AQP3 contributes to renal PC water permeability and this may be sex-dependent. Thus, acetylated AQP3 is a novel mechanism to maintain water homeostasis. R01DK128001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Comparing Effects of Tolvaptan and Instruction to Increase Water Consumption in ADPKD: Post Hoc Analysis of TEMPO 3:4.

In addition to decreasing water excretion and increasing urinary concentration, the antidiuretic hormone vasopressin plays a role in the pathophysiology of autosomal dominant polycystic kidney disease (ADPKD). It has been hypothesized that by suppressing vasopressin release, drinking large amounts of water might exert therapeutic effects in ADPKD similar to those of tolvaptan, an antagonist of the vasopressin type 2 receptor, but evidence is lacking. We analyzed data from tolvaptan clinical trials to evaluate relationships among water intake, urine osmolality (Uosm), and change in total kidney volume (TKV). Analysis of the TEMPO 3:4 clinical trial in which participants were randomized to tolvaptan or placebo and instructed to drink large amounts of water. The relationship between change in spot Uosm from baseline to Week 3 and change in TKV to Month 12 was assessed using linear regression modeling. Two short-term tolvaptan trials were analyzed to explore relationships between intermittent Uosm sampling and 24-hour Uosm suppression. With both tolvaptan and placebo (i.e., mandated high water intake alone), Uosm reduction at Week 3 was associated with reduction in TKV growth at Month 12. However, for the same decrease in spot Uosm, the corresponding reduction in TKV growth was greater for tolvaptan (for example, a -250 mOsm/kg reduction in Uosm at Week 3 was associated with a -1% change in TKV at Month 12 for tolvaptan versus +4.5% for placebo). In short-term trials, similar reductions in spot or trough Uosm values were achievable with tolvaptan and high water intake, but cumulative 24-hour suppression was greater with tolvaptan. This analysis supports a relationship between effects on Uosm and inhibition of disease progression by tolvaptan and high water intake alone. The findings further suggest that 24-hour Uosm measurement is superior to spot Uosm for assessing suppression of vasopressin activity by tolvaptan.

LRBA signalosomes activate vasopressin-induced AQP2 trafficking at recycling endosomes.

Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba-/- mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.

Ageing restructures the transcriptome of the hypothalamic supraoptic nucleus and alters the response to dehydration

Ageing is associated with altered neuroendocrine function. In the context of the hypothalamic supraoptic nucleus, which makes the antidiuretic hormone vasopressin, ageing alters acute responses to hyperosmotic cues, rendering the elderly more susceptible to dehydration. Chronically, vasopressin has been associated with numerous diseases of old age, including type 2 diabetes and metabolic syndrome. Bulk RNAseq transcriptome analysis has been used to catalogue the polyadenylated supraoptic nucleus transcriptomes of adult (3 months) and aged (18 months) rats in basal euhydrated and stimulated dehydrated conditions. Gene ontology and Weighted Correlation Network Analysis revealed that ageing is associated with alterations in the expression of extracellular matrix genes. Interestingly, whilst the transcriptomic response to dehydration is overall blunted in aged animals compared to adults, there is a specific enrichment of differentially expressed genes related to neurodegenerative processes in the aged cohort, suggesting that dehydration itself may provoke degenerative consequences in aged rats.

Identification of protein kinase A signalling molecules in renal collecting ducts.

Body water homeostasis is maintained by the correct balance between water intake and water loss through urine, faeces, sweat and breath. It is known that elevated circulating levels of the antidiuretic hormone vasopressin decrease urine volume to prevent excessive water loss from the body. Vasopressin/cAMP/protein kinase A (PKA) signalling is the canonical pathway in renal collecting ducts for phosphorylating aquaporin-2 (AQP2) water channels, which leads to the reabsorption of water from urine via AQP2. Although recent omics data have verified various downstream targets of PKA, crucial regulators that mediate PKA-induced AQP2 phosphorylation remain unknown, mainly because vasopressin is usually used to activate PKA as a positive control. Vasopressin is extremely potent and phosphorylates various PKA substrates non-specifically, making it difficult to narrow down the candidate mediators responsible for AQP2 phosphorylation. The intracellular localization of PKA is tightly regulated by its scaffold proteins, also known as A-kinase anchoring proteins (AKAPs). Furthermore, each AKAP has a target domain that determines its intracellular localization, enabling the creation of a local PKA signalling network. Although vasopressin activates most PKAs independently of their intracellular localization, some chemical compounds preferentially act on PKAs localized on AQP2-containing vesicles while simultaneously phosphorylating AQP2 and its surrounding PKA substrates. Immunoprecipitation with antibodies against phosphorylated PKA substrates followed by mass spectrometry analysis revealed that the PKA substrate in proximity to AQP2 was lipopolysaccharide-responsive and beige-like anchor (LRBA). Furthermore, Lrba knockout studies revealed that LRBA was required for vasopressin-induced AQP2 phosphorylation.

Natural Peptide Toxins as an Option for Renewed Treatment of Type 2 Vasopressin Receptor-Related Diseases.

The type 2 vasopressin receptor (V2R) is expressed in the kidneys, and it is the keystone of water homeostasis. Under the control of the antidiuretic hormone vasopressin, the V2R ensures vital functions, and any disturbance has dramatic consequences. Despite decades of research to develop drugs capable of activating or blocking V2R function to meet real medical needs, only one agonist and one antagonist are virtually used today. These two drugs cover only a small portion of patients' needs, leaving millions of patients without treatment. Natural peptide toxins known to act selectively and at low doses on their receptor target could offer new therapeutic options.

Fluid homeostasis induced by sodium-glucose cotransporter 2 inhibitors: novel insight for better cardio-renal outcomes in chronic kidney disease.

Hypertension in chronic kidney disease (CKD) patients is a risk factor for end-stage renal disease, cardiovascular events, and mortality. Thus, the prevention and appropriate management of hypertension in these patients are essential strategies for better cardio-renal outcomes. In this review, we show novel risk factors for hypertension with CKD, several promising prognostic markers and treatment for cardio-renal outcomes. Of note, the clinical use of sodium-glucose cotransporter 2 (SGLT2) inhibitors has recently expanded to non-diabetic patients with CKD and heart failure as well as diabetic patients. SGLT2 inhibitors have an antihypertensive effect, but are also associated with a low risk of hypotension. This unique mechanism of blood pressure regulation by SGLT2 inhibitors may partially depend on body fluid homeostasis, which is mediated by the autoregulation property between "accelerator" (diuretic action) and "brake" (increase in anti-diuretic hormone vasopressin and fluid intake). Mineralocorticoid receptor (MR) blockers are used in the treatment of essential hypertension and hyperaldosteronism. Recently, a new MR blocker, finerenone, has been launched as a treatment for CKD with type 2 diabetes. These advances in relation to hypertension in CKD may contribute to the reduction of renal and cardiovascular events.

Osmoadaptive GLP-1R signalling in hypothalamic neurones inhibits antidiuretic hormone synthesis and release

ObjectivesThe excessive release of the antidiuretic hormone vasopressin is implicated in many diseases including cardiovascular disease, diabetes, obesity, and metabolic syndrome. Once thought to be elevated as a consequence of diseases, data now supports a more causative role. We have previously identified CREB3L1 as a transcription factor that co-ordinates vasopressin synthesis and release in the hypothalamus. The objective here was to identify mechanisms orchestrated by CREB3L1 that co-ordinate vasopressin release. MethodsWe mined Creb3l1 knockdown SON RNA-seq data to identify downstream target genes. We proceeded to investigate the expression of these genes and associated pathways in the supraoptic nucleus of the hypothalamus in response to physiological and pharmacological stimulation. We used viruses to selectively knockdown gene expression in the supraoptic nucleus and assessed physiological and metabolic parameters. We adopted a phosphoproteomics strategy to investigate mechanisms that facilitate hormone release by the pituitary gland. ResultsWe discovered glucagon like peptide 1 receptor (Glp1r) as a downstream target gene and found increased expression in stimulated vasopressin neurones. Selective knockdown of supraoptic nucleus Glp1rs resulted in decreased food intake and body weight. Treatment with GLP-1R agonist liraglutide decreased vasopressin synthesis and release. Quantitative phosphoproteomics of the pituitary neurointermediate lobe revealed that liraglutide initiates hyperphosphorylation of presynapse active zone proteins that control vasopressin exocytosis. ConclusionIn summary, we show that GLP-1R signalling inhibits the vasopressin system. Our data advises that hydration status may influence the pharmacodynamics of GLP-1R agonists so should be considered in current therapeutic strategies.

Updates and Perspectives on Aquaporin-2 and Water Balance Disorders.

Ensuring the proper amount of water inside the body is essential for survival. One of the key factors in the maintenance of body water balance is water reabsorption in the collecting ducts of the kidney, a process that is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and impermeable to ions or other small molecules. Impairments of AQP2 result in various water balance disorders, including nephrogenic diabetes insipidus (NDI), which is a disease characterized by a massive loss of water through the kidney and consequent severe dehydration. Dysregulation of AQP2 is also a cause of water retention with hyponatremia in heart failure, hepatic cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Antidiuretic hormone vasopressin is an upstream regulator of AQP2. Its binding to the vasopressin V2 receptor promotes AQP2 targeting to the apical membrane and thus enables water reabsorption. Tolvaptan, a vasopressin V2 receptor antagonist, is effective and widely used for water retention with hyponatremia. However, there are no studies showing improvement in hard outcomes or long-term prognosis. A possible reason is that vasopressin receptors have many downstream effects other than AQP2 function. It is expected that the development of drugs that directly target AQP2 may result in increased treatment specificity and effectiveness for water balance disorders. This review summarizes recent progress in studies of AQP2 and drug development challenges for water balance disorders.

Urinary Aquaporin 2 as a Potential Indicator Predicting Tolvaptan Response in Patients With ADPKD

IntroductionTolvaptan is used to treat autosomal dominant polycystic kidney disease (ADPKD) because it inhibits binding of the antidiuretic hormone vasopressin to the vasopressin V2 receptor (V2R), which suppresses the insertion of preformed water channel aquaporin 2 (AQP2) molecules in the luminal membrane of the collecting duct cells.MethodsThis single-center, prospective observational cohort study investigated whether decreased AQP2 elimination in urine affects the renal prognosis of patients who received tolvaptan. We selected 92 patients with ADPKD who were administered tolvaptan in our hospital. We evaluated correlations between changes in urinary AQP2 (U-AQP2) and clinical parameters and the annual change in total kidney volume (TKV) and estimated glomerular filtration rate (eGFR) as renal prognostic factors using univariable and multivariable multiple regression analyses.ResultsThe observation period was 2.4 ± 1.5 years. U-AQP2 per milligram of urinary creatinine (U-AQP2/Cr) decreased from 67.8 ± 50.6 to 20.7 ± 15.1 fmol/mg urinary creatinine after 1 month of tolvaptan treatment. This initial change in U-AQP2/Cr was correlated with high baseline U-AQP2/Cr, low baseline eGFR, and a large initial change in eGFR (baseline to 1 month). The initial change in U-AQP2/Cr (baseline to 1 month) was strongly correlated with the annual change in TKV and eGFR in multivariable analysis.ConclusionInitial decrease in U-AQP2/Cr in the first month of treatment reflects the pharmacologic effect of tolvaptan and could be an indicator of renal prognosis during tolvaptan treatment.

High dietary salt amplifies osmoresponsiveness in vasopressin-releasing neurons

SUMMARYHigh dietary salt increases arterial pressure partly through activation of magnocellular neurosecretory cells (MNCVP) that secrete the antidiuretic and vasoconstrictor hormone vasopressin (VP) into the circulation. Here, we show that the intrinsic and synaptic excitation of MNCVP caused by hypertonicity are differentially potentiated in two models of salt-dependent hypertension in rats. One model combined salty chow with a chronic subpressor dose of angiotensin II (AngII-salt), the other involved replacing drinking water with 2% NaCl (salt loading, SL). In both models, we observed a significant increase in the quantal amplitude of EPSCs on MNCVP. However, model-specific changes were also observed. AngII-salt increased the probability of glutamate release by osmoreceptor afferents and increased overall excitatory network drive. In contrast, SL specifically increased membrane stiffness and the intrinsic osmosensitivity of MNCVP. These results reveal that dietary salt increases the excitability of MNCVP through effects on the cell-autonomous and synaptic osmoresponsiveness of MNCVP.

Unique Organization of Actin Cytoskeleton in Magnocellular Vasopressin Neurons in Normal Conditions and in Response to Salt-Loading.

Magnocellular neurosecretory cells (MNCs) are intrinsically osmosensitive and can be activated by increases in blood osmolality, triggering the release of antidiuretic hormone vasopressin (VP) to promote water retention. Hence, the activity of magnocellular VP neurons is one of the key elements contributing to the regulation of body fluid homeostasis in healthy organisms. Chronic exposure to high dietary salt leads to excessive activation of VP neurons, thereby elevating levels of circulating VP, which can cause increases in blood pressure contributing to salt-dependent hypertension. However, the molecular basis underlying high-salt diet-induced hyperactivation of magnocellular VP neurons remains not fully understood. Previous studies suggest that magnocellular neurosecretory neurons contain a subcortical layer of actin filaments and pharmacological stabilization of this actin network potentiates osmotically-induced activation of magnocellular neurons. Using super-resolution imaging in situ, we investigated the organization of the actin cytoskeleton in rat MNCs under normal physiological conditions and after a chronic increase in blood osmolality following 7 d of salt-loading (SL). We found that, in addition to the subcortical layer of actin filaments, magnocellular VP neurons are endowed with a unique network of cytoplasmic actin filaments throughout their somata. Moreover, we revealed that the density of both subcortical and cytoplasmic actin networks in magnocellular VP neurons is dramatically increased following SL. These results suggest that increased osmo-responsiveness of VP neurons following chronic exposure to high dietary salt may be mediated by the modulation of unique actin networks in magnocellular VP neurons, possibly contributing to elevated blood pressure in this condition.

Amyloid-like aggregation of provasopressin.

The antidiuretic hormone vasopressin is synthesized as a longer precursor protein. After folding in the endoplasmic reticulum (ER), provasopressin is transported through the secretory pathway, forms secretory granules in the trans-Golgi network (TGN), is processed, and finally secreted into the circulation. Mutations in provasopressin cause autosomal dominant diabetes insipidus. They prevent native protein folding and cause fibrillar, amyloid-like aggregation in the ER, which eventually results in cell death. Secretory granules of peptide hormones were proposed to constitute functional amyloids and thus might be the cause of amyloid formation of misfolded mutant protein in the ER. Indeed, the same two segments in the precursor-vasopressin and a C-terminal glycopeptide-were found to be responsible for pathological aggregation in the ER and physiological aggregation in granule formation in the TGN. Furthermore, even wild-type provasopressin tends to aggregate in the ER, but is controlled by ER-associated degradation. When essential components thereof, Sel1L or Hrd1, were inactivated, wild-type provasopressin accumulated as fibrillar aggregates in vasopressinergic neurons in mice, causing diabetes insipidus. Evolution of amyloidogenic sequences for granule formation thus made provasopressin dependent on ER quality control mechanisms. These principles may similarly apply to other peptide hormones.

A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging.

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

Site-1 protease-derived soluble (pro)renin receptor targets vasopressin receptor 2 to enhance urine concentrating capability.

The antidiuretic hormone vasopressin (AVP), acting through its type 2 receptor (V2R) in the collecting duct (CD), critically controls urine concentrating capability. Here, we report that site-1 protease-derived (S1P-derived) soluble (pro)renin receptor (sPRR) participates in regulation of fluid homeostasis via targeting V2R. In cultured inner medullary collecting duct (IMCD) cells, AVP-induced V2R expression was blunted by a PRR antagonist, PRO20; a PRR-neutralizing antibody; or a S1P inhibitor, PF-429242. In parallel, sPRR release was increased by AVP and reduced by PF-429242. Administration of histidine-tagged sPRR, sPRR-His, stimulated V2R expression and also reversed the inhibitory effect of PF-429242 on the expression induced by AVP. PF-429242 treatment in C57/BL6 mice impaired urine concentrating capability, which was rescued by sPRR-His. This observation was recapitulated in mice with renal tubule-specific deletion of S1P. During the pharmacological or genetic manipulation of S1P alone or in combination with sPRR-His, the changes in urine concentration were paralleled with renal expression of V2R and aquaporin-2 (AQP2). Together, these results support that S1P-derived sPRR exerts a key role in determining renal V2R expression and, thus, urine concentrating capability.

Combining Desmopressin and Docetaxel for the Treatment of Castration-Resistant Prostate Cancer in an Orthotopic Model.

Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in breast cancer models. Docetaxel is a chemotherapy agent for castrate-resistant prostate cancer (CRPC). In this study, the ability of CRPC cells to grow and develop in vivo tumors in an animal model was evaluated, in order to investigate the anti-tumor effect of desmopressin in combination with docetaxel. The CRPC cell line PC3 was used for orthotopic inoculation in male athymic nude mice. The mice were randomly assigned to one of the four treatment groups: Control, docetaxel, desmopressin or combination therapy. Following the last treatment, tumors were excised and measured. Blood samples were processed for CTC analysis. Docetaxel treatment resulted in a significant reduction in tumor volume compared to control. The combination therapy resulted in even more significant reduction (31.2%) in tumor volume. There was a complete absence of CTCs in the combination group. Our pilot study demonstrated an enhanced efficacy of docetaxel-based therapy in combination with desmopressin.

Soluble (pro)renin receptor as a potential therapy for diabetes insipidus.

The antidiuretic hormone vasopressin (VP) is produced by the hypothalamus and is stored and secreted from the posterior pituitary. VP acts via VP type 2 receptors (V2Rs) on the basolateral membrane of principal cells of the collecting duct (CD) to regulate fluid permeability. The VP-evoked endocrine pathway is essential in determining urine concentrating capability. For example, a defect in any component of the VP signaling pathway can result in polyuria, polydipsia, and hypotonic urine, collectively termed diabetes insipidus (DI). A lack of VP production precipitates central diabetes insipidus (CDI), which can be managed effectively by VP supplementation. A majority of cases of nephrogenic diabetes insipidus (NDI) result from V2R mutations that impair receptor sensitivity. No specific therapy is currently available for management of NDI. Evidence is evolving that (pro)renin receptor (PRR), a newly identified member of the renin-angiotensin system, is capable of regulating VP production and action. As such, PRR should be considered strongly as a therapeutic target for treating CDI and NDI. The current review will summarize recent advances in understanding the physiology of renal and central PRR as it relates to the two types of DI.