A novel AVPR2 gene mutation in a Chinese pedigree with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare genetic disorder primarily associated with mutations in the arginine vasopressin receptor 2 (AVPR2) gene or the aquaporin 2 (AQP2) gene, resulting in impaired water reabsorption in the renal tubules. This report describes a case of a young male patient with NDI from China with a history of polydipsia and polyuria for over 15 years. Laboratory examinations of the proband indicated low urine-specific gravity and osmolality. Urologic ultrasound revealed severe bilateral hydronephrosis in both kidneys, bilateral dilatation of the ureters, roughness of the bladder wall, and the formation of muscle trabeculae. The diagnosis of diabetes insipidus was confirmed by water deprivation tests. The administration of posterior pituitary hormone did not alter urine-specific gravity, and osmolality remained at a low level (<300 mOsm/kg). Based on these findings, and the genetic tests of the proband and his parents were performed. A missense mutation (c.616 G>C) in exon 3 of the AVPR2 gene of the proband was found, caused by the substitution of amino acid valine to leucine at position 206 [p.Val206Leu], which was a hemizygous mutation and consistent with X-chromosome recessive inheritance. The administration of oral hydrochlorothiazide improves the symptoms of polydipsia and polyuria in the proband. This novel AVPR2 gene mutation may be the main cause of NDI in this family, which induces a functional defect in AVPR2, and leads to reduced tubular reabsorption of water.

A better understanding of basic science may help our management of LUTS/LUTD in older persons with nocturnal polyuria and nocturia: ICI-RS 2024.

AIMS: To discuss the role of autocrine/paracrine signaling of urothelial arginine vasopressin (AVP) on mammalian bladder capacities and micturition thresholds, impact of distension on water/urea reabsorption from the bladder, review of the literature to better characterize the central/peripheral effects of AVP, desmopressin (dAVP) toxicity, and urine biomarkers of nocturia. METHODS: This review summarizes discussions during an International Consultation on Incontinence-Research Society 2024 think tank with respect to the role of urothelial AVP in aged individuals with nocturnal polyuria, impact of solute and water reabsorption by the bladder on uninterrupted sleep, central effects of AVP, pharmacological basis of dAVP toxicity, and biomarkers in nocturia/lower urinary tract dysfunction (LUTD) with neurological diseases. RESULTS: Consensus recognized AVP function and pathways in the central nervous system (CNS), pre-proAVP localized using immunohistochemistry in bladder sections from adult/aged noncancerous human punch biopsies and rodent bladder sections is likely to accelerate the systemic uptake of water and urea from the bladder of anesthetized mice instilled with 3H-water and 14C-urea. Mechanisms for charged and uncharged solutes and water transport across the bladder, mechanism of dAVP toxicity, and utility of urine biomarkers in those with neurological diseases/nocturia were determined from literature reviews. CONCLUSION: Pre-proAVP is present in human/rodent bladders and may be involved in water reabsorption from bladder that prevents the sensation of fullness for uninterrupted sleep in healthy adults. The mechanism of action of AVP in the CNS was discussed, as was electrolyte/water transport across the bladder, the basis for dAVP toxicity, and feasibility of urine biomarkers to identify nocturia/LUTD with neurological diseases.

Genetic analysis of nephrogenic diabetes insipidus patients: A study on the Iranian population.

INTRODUCTION: Nephrogenic diabetes insipidus (NDI) is a rare genetic disease that causes water imbalance. The kidneys play a crucial role in regulating body fluids by controlling water balance through urine excretion. This highlights their essential function in managing the body's water levels, but individuals with NDI may have excess urine production (polyuria), that leads to excessive thirst (polydipsia). Untreated affected individuals may exhibit poor feeding and failure to thrive. This disease is caused by mutations in the AVPR2 and the AQP2 genes which have the X-linked and autosomal recessive/dominant inheritance, respectively. Both of these genes are expressed in the kidney. METHODS: Twelve Iranian patients from 10 consanguineous families were studied in this project. DNA was extracted from the whole blood samples of the patients and their parents. All coding exons and exon-intron boundaries of the AVPR2 and AQP2 genes were sequenced in the affected individuals, and the identified variants were investigated in the parents. All variants were analyzed according to the ACMG (American College of Medical Genetics and Genomics) guidelines. RESULTS: In this study, 6 different mutations were identified in the patients, including 5 in the AQP2 gene (c.439G>A, c.538G>A, c.140C>T, c.450T>A, and the novel c.668T>C) and 1 in the AVPR2 gene (c.337C>T) in the present study. DISCUSSION: As expected, all the detected mutations in this study were missense. According to the ACMG guideline, the identified mutations were categorized as pathogenic or likely pathogenic. Unlike previous studies which showed more than 90% of mutations were in the AVPR2 gene, and only less than 10% of the mutations were in the AQP2 gene, it was found that more than 90% of our identified mutations located in the AQP2 gene, and only one mutation was observed in the AVPR2 gene, which seems it may be a result of the high rate of consanguineous marriages in the Iranian population. We observed genotype-phenotype correlation in some of our affected individuals, and some of the mutations were observed in unrelated families from same ethnicity which could be suggestive of a founder mutation.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Efficacy and safety of V1a receptor antagonists in autism spectrum disorder: A meta-analysis.

This meta-analysis has evaluated the efficacy and safety of V1a receptor antagonists in ASD compared to placebo. The reviewers extracted data from four relevant clinical trials after a literature search on databases and clinical trial registries. Quality assessment was done using the risk of bias assessment tool, and the random-effects model was used to estimate effect size. Subgroup analysis, meta-regression and sensitivity analysis were done. PRISMA guidelines were followed in the selection, analysis and reporting of findings. V1a receptor antagonists did not reduce Vineland II Adaptive behaviour composite score significantly (SMD: 0.14; 95% CI: -0.06-0.35; p = 0.16; PI: -0.44-0.73), communication domain subscale score and socialization domain subscale score. The change in daily living skills domain subscale score was significant and favourable for V1a receptor antagonists (SMD: 0.15; 95% CI: 0.03-0.26; p = 0.01). The subgroup analysis revealed a significant improvement in Vineland II Adaptive behaviour composite score with doses <10 mg (SMD: 0.45; 95% CI: 0.11-0.78; p = 0.009). Meta-regression does not show a significant association between SMD of ASD symptom score reduction with the duration and dose of V1a receptor antagonist therapy. Treatment-emergent adverse effects were not serious and dose dependent. Low doses (<10 mg) of V1a receptor antagonist may be effective in reducing the core symptoms of ASD compared to placebo; however, future active-controlled clinical trials are necessary to generate conclusive evidence.

Influence of vasopressin receptor antagonists on triple-whammy acute kidney injury: A VigiBase analysis.

Although diuretics play an important role in triple-whammy acute kidney injury (AKI), it is unclear whether the type of diuretic influences the risk of triple-whammy AKI. The aim of this study was to evaluate whether vasopressin receptor antagonists affect triple-whammy AKI. This cross-sectional study used disproportionality analysis of VigiBase data to assess the risk of AKI with various diuretics. Although multiple logistic regression analysis showed that aldosterone antagonists (odds ratio [OR] 2.19, 95% CI 2.01-2.37), loop diuretics (OR 4.40, 95% CI 4.07-4.76) and thiazide diuretics (OR 1.98, 95% CI 1.83-2.15) increased the risk of AKI in patients who received non-steroidal anti-inflammatory drugs (NSAIDs) and renin-angiotensin system inhibitors (RASi), vasopressin receptor antagonists did not increase the risk of AKI in those patients. Vasopressin receptor antagonists might not influence the development of triple-whammy AKI.

Safety and Efficacy of Vaptans in the Treatment of Hyponatremia from Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH): A Systematic Review and Meta-Analysis.

The utilization of vasopressin receptor antagonists, known as vaptans, in the management of hyponatremia among patients afflicted with the syndrome of inappropriate antidiuretic hormone (SIADH) remains a contentious subject. This meta-analysis aimed to evaluate the safety and efficacy of vaptans for treating chronic hyponatremia in adult SIADH patients. Clinical trials and observational studies were identified by a systematic search using MEDLINE, EMBASE, and Cochrane Database from inception through September 2022. The inclusion criteria were the studies that reported vaptans' safety or efficacy outcomes compared to placebo or standard therapies. The study protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; CRD 42022357307). Five studies were identified, comprising three RCTs and two cohort studies, enrolling a total of 1840 participants. Regarding short-term efficacy on days 4-5, vaptans exhibited a significant increase in serum sodium concentration from the baseline in comparison to the control group, with a weighted mean difference of 4.77 mmol/L (95% CI, 3.57, 5.96; I2 = 34%). In terms of safety outcomes, the pooled incidence rates of overcorrection were 13.1% (95% CI 4.3, 33.6; I2 = 92%) in the vaptans group and 3.3% (95% CI 1.6, 6.6; I2 = 27%) in the control group. Despite the higher correction rate linked to vaptans, with an OR of 5.72 (95% CI 3.38, 9.70; I2 = 0%), no cases of osmotic demyelination syndrome were observed. Our meta-analysis comprehensively summarizes the efficacy and effect size of vaptans in managing SIADH. While vaptans effectively raise the serum sodium concentration compared to placebo/fluid restriction, clinicians should exercise caution regarding the potential for overcorrection.

Target immobilization on glass microfiber membranes as a label-free strategy for hit identification.

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V2R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V2R were immobilized on the surface of a glass microfiber (GMF) coated with O6-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V2R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V2R ligand treated with SNAP-V2R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the Ki value determined by displacement assays. Furthermore, a Ki prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V2R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V2R@GMF-BG was incubated with the total extract, the ligand that binds to V2R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V2R antagonist. This V2R-immobilized GMF platform can help determine the affinity of V2R-binding hit compounds and screen the compounds efficiently and accurately.

Effects of Vasopressin Receptor Agonists during the Resuscitation of Hemorrhagic Shock: A Systematic Review and Meta-Analysis of Experimental and Clinical Studies.

BACKGROUND: The clinical impact of vasopressin in hemorrhagic shock remains largely unknown. OBJECTIVE: This systematic review and meta-analysis was designed to investigate the effects of vasopressin receptor agonists during the resuscitation of hemorrhagic shock. METHODS: A systematic search of PubMed (MEDLINE), Scopus, and PubMed Central was conducted for relevant articles. Experimental (animal) and clinical studies were included. The primary objective was to investigate the correlation of vasopressin receptor agonist use with mortality and various hemodynamic parameters. RESULTS: Data extraction was possible in thirteen animal studies and two clinical studies. Differences in risk of mortality between patients who received a vasopressin receptor agonist were not statistically significant when compared to those who were not treated with such agents [RR (95% CI): 1.17 (0.67, 2.08); p = 0.562; I2 = 50%]. The available data were insufficient to conduct a meta-analysis assessing the effect of vasopressin receptor agonists on hemodynamics. Drawing safe conclusions from animal studies was challenging, due to significant heterogeneity in terms of species and dosage of vasopressin receptor agonists among studies. CONCLUSIONS: Differences in risk of mortality between patients who received a vasopressin receptor agonist were not statistically significant when compared to those who were not treated with such agents after hemorrhagic shock. More data are needed to deduce certain conclusions.

Association between arginine vasopressin receptor 1A (AVPR1A) polymorphism and inequity aversion.

Although numerous studies have focused on brain functions related to inequity aversion, few have examined its genetic basis. Here, we show the association between estimated inequity aversion and polymorphisms in three genes associated with human sociality. Non-student adult participants took part in five economic game experiments on different days. Disadvantageous inequity aversion (DIA) and advantageous inequity aversion (AIA) were calculated from behavioural responses using Bayesian estimation. We investigated the association between genetic polymorphisms in the oxytocin receptor (OXTR rs53576), arginine vasopressin receptor 1A (AVPR1A RS3) and opioid receptor mu 1 (OPRM1 rs1799971) and inequity aversion. Regarding AVPR1A RS3, participants with the SS genotype had higher AIA than those with the SL or LL genotypes, but no association was found for DIA. Moreover, we observed no aversion associations for OXTR rs53576 or OPRM1 rs1799971. The results suggest that AVPR1A plays an important role in aversion when one's own gain is greater than that of others. Our findings may provide a solid theoretical basis for future studies on the relationship between genetic polymorphisms and inequity aversion.

Assessment of Tissue Expression of the Oxytocin-Vasopressin Pathway in the Placenta of Women with a First-Episode Psychosis during Pregnancy.

Psychosis refers to a mental health condition characterized by a loss of touch with reality, comprising delusions, hallucinations, disorganized thought, disorganized behavior, catatonia, and negative symptoms. A first-episode psychosis (FEP) is a rare condition that can trigger adverse outcomes both for the mother and newborn. Previously, we demonstrated the existence of histopathological changes in the placenta of pregnant women who suffer an FEP in pregnancy. Altered levels of oxytocin (OXT) and vasopressin (AVP) have been detected in patients who manifested an FEP, whereas abnormal placental expression of these hormones and their receptors (OXTR and AVPR1A) has been proven in different obstetric complications. However, the precise role and expression of these components in the placenta of women after an FEP have not been studied yet. Thus, the purpose of the present study was to analyze the gene and protein expression, using RT-qPCR and immunohistochemistry (IHC), of OXT, OXTR, AVP, and AVPR1a in the placental tissue of pregnant women after an FEP in comparison to pregnant women without any health complication (HC-PW). Our results showed increased gene and protein expression of OXT, AVP, OXTR, and AVPR1A in the placental tissue of pregnant women who suffer an FEP. Therefore, our study suggests that an FEP during pregnancy may be associated with an abnormal paracrine/endocrine activity of the placenta, which can negatively affect the maternofetal wellbeing. Nevertheless, additional research is required to validate our findings and ascertain any potential implications of the observed alterations.

Bringing GPCR Structural Biology to Medical Applications: Insights from Both V2 Vasopressin and Mu-Opioid Receptors.

G-protein coupled receptors (GPCRs) are versatile signaling proteins that regulate key physiological processes in response to a wide variety of extracellular stimuli. The last decade has seen a revolution in the structural biology of clinically important GPCRs. Indeed, the improvement in molecular and biochemical methods to study GPCRs and their transducer complexes, together with advances in cryo-electron microscopy, NMR development, and progress in molecular dynamic simulations, have led to a better understanding of their regulation by ligands of different efficacy and bias. This has also renewed a great interest in GPCR drug discovery, such as finding biased ligands that can either promote or not promote specific regulations. In this review, we focus on two therapeutically relevant GPCR targets, the V2 vasopressin receptor (V2R) and the mu-opioid receptor (µOR), to shed light on the recent structural biology studies and show the impact of this integrative approach on the determination of new potential clinical effective compounds.

V2 vasopressin receptor mutations: future personalized therapy based on individual molecular biology.

The diluting and concentrating function of the kidney plays a crucial role in regulating the water homeostasis of the body. This function is regulated by the antidiuretic hormone, arginine vasopressin through the type 2 vasopressin receptor (V2R), allowing the body to adapt to periods of water load or water restriction. Loss-of-function mutations of the V2R cause X-linked nephrogenic diabetes insipidus (XNDI), which is characterized by polyuria, polydipsia, and hyposthenuria. Gain-of-function mutations of the V2R lead to nephrogenic syndrome of inappropriate antidiuresis disease (NSIAD), which results in hyponatremia. Various mechanisms may be responsible for the impaired receptor functions, and this review provides an overview of recent findings about the potential therapeutic interventions in the light of the current experimental data.

The ameliorative effect of midazolam on empathy-like behavior in old rats.

Although studies suggest that cognitive functions in the elderly are impaired, elderly people tend to be more successful and wiser in solving emotional problems. In empathy-like behavior models, the observer rat rescues the distressed cage mate by displaying emotional and cognitive ability. The aim of the study was to investigate the changes in empathy-like behavior in older rats in comparison to adult rats. In addition, we wanted to determine the effects of alterations in neurochemicals (such as corticosterone, oxytocin, vasopressin, and their receptor levels) and emotional situations on this behavior. In our study, we initially completed empathy-like behavior tests and emotional tests (open field, elevated plus maze) and performed neurochemical examinations in the serum and brain tissues. In the second step of research, we applied a midazolam (benzodiazepine) treatment to examine the effect of anxiety on empathy-like behavior. In the old rats, we observed that empathy-like behavior deteriorated, and anxiety signs were more pronounced. We detected a positive correlation between the latency in empathy-like behavior and corticosterone levels and v1b receptor levels. The midazolam effect on empathy-like behavior was attenuated by flumazenil (a benzodiazepine receptor antagonist). The recordings of ultrasonic vocalization showed frequencies around 50 kHz emitted by the observer and this was associated with the expectation of social contact. Our results state that compared to adult rats, old rats were more concerned and failed during empathy-like behavior. Midazolam may improve this behavior by anxiolysis.

Prenatal ketamine exposure impairs prepulse inhibition via arginine vasopressin receptor 1A-mediated GABAergic neuronal dysfunction in the striatum.

Ketamine is a widely used anesthetic with N-methyl-D-aspartate (NMDA) receptor antagonism. Exposure to ketamine and NMDA receptor antagonists may induce psychosis. However, the mechanism underlying the effects of ketamine on the immature brain remains unclear. In this study, NMDA receptor antagonists, ketamine and methoxetamine, were administered to pregnant F344 rats (E17). These regimens induce psychosis-like behaviors in the offspring, such as hyperlocomotion induced by MK-801, a non-competitive NMDA receptor antagonist. We also observed that prepulse inhibition (PPI) was significantly reduced. Interestingly, ketamine administration increased the arginine vasopressin receptor 1A (Avpr1a) expression levels in the striatum of offspring with abnormal behaviors. Methoxetamine, another NMDA receptor antagonist, also showed similar results. In addition, we demonstrated a viral vector-induced Avpr1a overexpression in the striatum-inhibited PPI. In the striatum of offspring, ketamine or methoxetamine treatment increased glutamate decarboxylase 67 (GAD67) and δ-aminobutyric acid (GABA) levels. These results show that prenatal NMDA receptor antagonist treatment induces GABAergic neuronal dysfunction and abnormalities in sensorimotor gating via regulating Avpr1a expression in the striatum.

Novel signalling pathways in nephrogenic syndrome of inappropriate antidiuresis: functional implication of site-specific AQP2 phosphorylation.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of Rho-associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain-of-function V2R-R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. KEY POINTS: Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R-F229V) and the other vaptan resistant (V2R-R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R-R137L/C mutants, disruption of the AQP2-S269 phosphorylation site caused the loss of the gain-of-function phenotype, which, in contrast, was retained in V2R-F229V-expressing cells. Cells expressing the V2R-F229V mutant were instead sensitive to disruption of the AQP2-S256 phosphorylation site. The serine/threonine kinase Rho-associated kinase (ROCK) was found to be involved in AQP2-S269 phosphorylation downstream of the V2R-R137L/C mutants. These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis.

Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2.

mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3'- and 5'-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies.

Genetic basis of nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus is defined as an inability to concentrate urine due to a complete or partial alteration of the renal tubular response to arginine vasopressin hormone, resulting in excessive diluted urine excretion. Hereditary forms are caused by molecular defects in the genes encoding either of the two main renal effectors of the arginine vasopressin pathway: the AVPR2 gene, which encodes for the type 2 vasopressin receptor, or the AQP2 gene, which encodes for the water channel aquaporin-2. About 90% of cases of nephrogenic diabetes insipidus result from loss-of-function variants in the AVPR2 gene, which are inherited in a X-linked recessive manner. The remaining 10% of cases result from loss-of-function variants in the AQP2 gene, which can be inherited in either a recessive or a dominant manner. The main symptoms of the disease are polyuria, chronic dehydration and hypernatremia. These symptoms usually occur in the first year of life, although some patients present later. Diagnosis is based on abnormal response in urinary osmolality after water restriction and/or administration of exogenous vasopressin. Treatment involves ensuring adequate water intake on demand, possibly combined with thiazide diuretics, non-steroidal anti-inflammatory drugs, and a low-salt and protein diet. In this review, we provide an update on current understanding of the molecular basis of inherited nephrogenic insipidus diabetes.

Data resource: vasopressin-regulated protein phosphorylation sites in the collecting duct.

Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.