Vasopressin Receptor Type Research Articles

Molecular mechanism of β-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin pre-activation. We compare the conformational changes of β-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and βXX to transform β-arrestin-2 into the pre-active state.

Roles of the gate loop in β-arrestin-1 conformational dynamics and phosphorylated receptor interaction

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using β-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of β-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.

The β3-AR agonist BRL37344 ameliorates the main symptoms of X-linked nephrogenic diabetes insipidus in the mouse model of the disease.

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the β3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the β3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the β3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human β3-AR agonists might represent an effective possible treatment strategy for X-NDI.

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.

β-Arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

The vasopressin type 2 receptor (V2R) is an essential G protein-coupled receptor (GPCR) in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gαs and Gαq/11, which is followed by robust recruitment of β-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the β-arrestin association with the V2R does not terminate Gαs activation, and thus, Gαs-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/β-arrestin and promote endosomal G protein signaling is not restricted to Gαs, but also involves Gαq/11. Furthermore, our data imply that β-arrestins potentiate Gαs/Gαq/11 activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of β-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both β-arrestin-dependent and -independent manners.

β-Arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor

The vasopressin type 2 receptor (V2R) is an essential G protein-coupled receptor (GPCR) in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gαs and Gαq/11, which is followed by robust recruitment of β-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the β-arrestin association with the V2R does not terminate Gαs activation, and thus, Gαs-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/β-arrestin and promote endosomal G protein signaling is not restricted to Gαs, but also involves Gαq/11. Furthermore, our data imply that β-arrestins potentiate Gαs/Gαq/11 activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of β-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both β-arrestin-dependent and -independent manners.

Aquaporins in Edema.

One of the most prevalent indications of water-electrolyte imbalance is edema. Aquaporins (AQPs) are a protein family that can function as water channels. Osmoregulation and body water homeostasis are dependent on the regulation of AQPs. Human kidneys contain nine AQPs, five of which have been demonstrated to have a role in body water balance: AQP1, AQP2, AQP3, AQP4, and AQP7. Water imbalance is connected with AQP dysfunction. Hyponatremia with elevated AQP levels can accompany edema, which can be caused by disorders with low effective circulating blood volume and systemic vasodilation, such as congestive heart failure (CHF), hepatic cirrhosis, or the syndrome of incorrect antidiuretic hormone secretion (SIADH). In CHF, upregulation of AQP2 expression and targeting is critical for water retention. AQP2 is also involved in aberrant water retention and the formation of ascites in cirrhosis of the liver. Furthermore, water retention and hyponatremia in SIADH are caused by increased expression of AQP2 in the collecting duct. Fluid restriction, demeclocycline, and vasopressin type-2 receptor antagonists are widely utilized to treat edema. The relationship between AQPs and edema is discussed in this chapter.

Genetic deletion of the nuclear factor of activated T cells 5 in collecting duct principal cells causes nephrogenic diabetes insipidus.

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8h water restriction, NFAT5PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI.

Natural phenylethanoid glycosides diuretics derived from Lagopsis supina: Biological activity, mechanism, molecular docking, and structure-activity relationship

Aquaporins (AQPs) and vasopressin type 2 receptor (V2R) play a crucial role in urine excretion and are widely used to explore novel diuretics. In this study, three phenylpropanoids including stachysoside A (L1), acteoside (L2), and glucopyranosyl (1 → 6) martynoside (L3) were isolated from Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. Their diuretic activity, mechanism, molecular docking, and structure-activity relationships were explored. The results suggest that L1, L2, and L3 exhibit acute (6 h) and prolonged (6 d) activities including increased urinary excretion volume, diuretic action, and diuretic activity, without affecting the urinary pH and minor altering the electrolyte balance in saline-loaded rats. Further, L1, L2, and L3 significantly reduced the levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), and aldosterone (ALD), AQPs 1–4 and 7, and V2R, and remarkably elevated the atriopeptin (ANP) level. Besides, L1, L2, and L3 obviously suppressed mRNA and protein levels of AQPs 1–4 and 7, and V2R. The hypothetical binding modes of L1, L2, and L3 with these proteins were determined by molecular docking, and a tight structure-activity relationship was also proposed. Collectively, L1, L2, and L3 represent three natively novel phenylethanoid glycoside diuretics, which inhibit AQP and V2R-mediated molecular mechanisms. They are superior to furosemide as long-term diuretics.

Vasopressin Receptor Type-2 Mediated Signaling in Renal Cell Carcinoma Stimulates Stromal Fibroblast Activation.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

The V2 receptor antagonist tolvaptan counteracts proliferation and invasivity in human cancer cells

PurposeHyponatremia, the most frequent electrolyte alteration in clinical practice, has been associated with a worse prognosis in cancer patients. On the other hand, a better outcome has been related to serum sodium normalization. In vitro studies have shown that low extracellular sodium promotes cancer cells proliferation and invasiveness. Tolvaptan, a selective vasopressin receptor type 2 (V2) antagonist, has been effectively used in the last decade for the treatment of hyponatremia secondary to the Syndrome of Inappropriate Antidiuresis. A few in vitro data suggested a direct role of tolvaptan in counteracting cancer progression, so far. The aim of this study was to evaluate the effect and the mechanism of action of tolvaptan in cell lines from different tumours [i.e. colon cancer (HCT-8), hepatocarcinoma (HepG2), neuroblastoma (SK-N-AS)].Methods and resultsFirst, we showed that these cell lines express the V2 receptor. Tolvaptan significantly reduced cell proliferation with an IC50 in the micromolar range. Accordingly, reduced levels of cAMP, of the catalytic α subunit of PKA, and a reduced pAKT/AKT ratio were observed. Tolvaptan effectively inhibited cell cycle progression, whereas it induced apoptotis. Furthermore, it reduced cell invasiveness. In particular, anchorage-independent growth and the activity of collagenases type IV were blunted in the three cell lines. Accordingly, tolvaptan counteracted the RhoA/ROCK1–2 pathway, which has a pivotal role in regulating cell movement.ConclusionsOverall, these findings indicate that tolvaptan effectively inhibits tumour progression in vitro. Further studies should clarify whether the V2 receptor might be considered a possible target in anti-cancer strategies in the future.

Altered arginine vasopressin-cyclic AMP-aquaporin 2 pathway in patients with chronic kidney disease.

In the collecting ducts of the kidney, arginine vasopressin (AVP), cyclic adenosine monophosphate (cAMP), and aquaporin 2 (AQP2) play a pivotal role in maintaining fluid volume and serum osmolality in humans. However, their association among those with chronic kidney disease (CKD) remains uncertain. We prospectively included the out-patients with CKD and measured osmolality-related biomarkers including plasma AVP, urine cAMP, urine AQP2, and urine osmolality levels. Association among these parameters at each CKD stage was investigated. A total of 121 patients were included (median age 71years old [61-78], 89 men, estimated glomerular filtration ratio 28.6 [16.4-45.3] mL/min/1.73m2). Serum osmolality increased as CKD progression, accompanying incremental plasma AVP levels, whereas urine cAMP, urine AQP2, and urine osmolality decreased as CKD progression. At advanced CKD stage, urine cAMP remained low irrespective of the AVP stimulation, whereas urine cAMP levels varied according to the levels of plasma AVP at less advanced CKD stage. The associations between urine cAMP and urine AQP2 and between urine AQP2 and urine osmolality remained preserved irrespective of the CKD stages. Vasopressin type-2 receptor seems to be particularly impaired in patients with advanced CKD, whereas the signal cascade of the downstream of vasopressin type-2 receptor is relatively preserved. Urine cAMP might be a promising marker to estimate the residual function of the collecting duct.

Apelin and Vasopressin: The Yin and Yang of Water Balance.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling body fluid homeostasis and cardiovascular functions. Experimental data performed in rodents have shown that apelin has an aquaretic effect via its central and renal actions. In the brain, apelin inhibits the phasic electrical activity of vasopressinergic neurons and the release of vasopressin from the posterior pituitary into the bloodstream and in the kidney, apelin regulates renal microcirculation and counteracts in the collecting duct, the antidiuretic effect of vasopressin occurring via the vasopressin receptor type 2. In humans and rodents, if plasma osmolality is increased by hypertonic saline infusion/water deprivation or decreased by water loading, plasma vasopressin and apelin are conversely regulated to maintain body fluid homeostasis. In patients with the syndrome of inappropriate antidiuresis, in which vasopressin hypersecretion leads to hyponatremia, the balance between apelin and vasopressin is significantly altered. In order to re-establish the correct balance, a metabolically stable apelin-17 analog, LIT01-196, was developed, to overcome the problem of the very short half-life (in the minute range) of apelin in vivo. In a rat experimental model of vasopressin-induced hyponatremia, subcutaneously (s.c.) administered LIT01-196 blocks the antidiuretic effect of vasopressin and the vasopressin-induced increase in urinary osmolality, and induces a progressive improvement in hyponatremia, suggesting that apelin receptor activation constitutes an original approach for hyponatremia treatment.

MAGED2 controls vasopressin-induced aquaporin-2 expression in collecting duct cells

Mutations in the Melanoma-Associated Antigen D2 (MAGED2) cause antenatal Bartter syndrome type 5 (BARTS5). This rare disease is characterized by perinatal loss of urinary concentration capability and large urine volumes. The underlying molecular mechanisms of this disease are largely unclear. Here, we study the effect of MAGED2 knockdown on kidney cell cultures using proteomic and phosphoproteomic analyses. In HEK293T cells, MAGED2 knockdown induces prominent changes in protein phosphorylation rather than changes in protein abundance. MAGED2 is expressed in mouse embryonic kidneys and its expression declines during development. MAGED2 interacts with G-protein alpha subunit (GNAS), suggesting a role in G-protein coupled receptors (GPCR) signalling. In kidney collecting duct cell lines, Maged2 knockdown subtly modulated vasopressin type 2 receptor (V2R)-induced cAMP-generation kinetics, rewired phosphorylation-dependent signalling, and phosphorylation of CREB. Maged2 knockdown resulted in a large increase in aquaporin-2 abundance during long-term V2R activation. The increase in aquaporin-2 protein was mediated transcriptionally. Taken together, we link MAGED2 function to cellular signalling as a desensitizer of V2R-induced aquaporin-2 expression. SignificanceIn most forms of Bartter Syndrome, the underlying cause of the disease is well understood. In contrast, the role of MAGED2 mutations in a newly discovered form of Bartter Syndrome (BARTS5) is unknown. In our manuscript we could show that MAGED2 modulates vasopressin-induced protein and phosphorylation patterns in kidney cells, providing a broad basis for further studies of MAGED2 function in development and disease.

β3 adrenergic receptor as potential therapeutic target in ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) disrupts renal parenchyma through progressive expansion of fluid‐filled cysts. The only approved pharmacotherapy for ADKPD involves the blockade of the vasopressin type 2 receptor (V2R). V2R is a GPCR expressed by a subset of renal tubular cells and whose activation stimulates cyclic AMP (cAMP) accumulation, which is a major driver of cyst growth. The β3‐adrenergic receptor (β3‐AR) is a GPCR expressed in most segments of the murine nephron, where it modulates cAMP production. Since sympathetic nerve activity, which leads to activation of the β3‐AR, is elevated in patients affected by ADPKD, we hypothesize that β3‐AR might constitute a novel therapeutic target. We find that administration of the selective β3‐AR antagonist SR59230A to an ADPKD mouse model (Pkd1fl/fl;Pax8rtTA;TetO‐Cre) decreases cAMP levels, producing a significant reduction in kidney/body weight ratio and a partial improvement in kidney function. Furthermore, cystic mice show significantly higher β3‐AR levels than healthy controls, suggesting a correlation between receptor expression and disease development. Finally, β3‐AR is expressed in human renal tissue and localizes to cyst‐lining epithelial cells in patients. Thus, β3‐AR is a potentially interesting target for the development of new treatments for ADPKD.

Peak Lag Between Plasma Vasopressin and Urine Aquaporin-2 Following Cardiac Surgery.

Tolvaptan, a vasopressin type-2 receptor antagonist, is utilized to ameliorate fluid retention following cardiac surgery. However, the optimal timing of tolvaptan administration considering novel biomarkers remains unknown. We prospectively included patients who underwent cardiac surgery between 2016 and 2020. We measured perioperative trends of free water reabsorption mediators including plasma arginine vasopressin and urine aquaporin-2. A total of 20 patients (68 [60, 75] years old, 18 men) were included. Urine volume decreased gradually after the initial 3 hours following cardiac surgery. The plasma arginine vasopressin level increased significantly with a peak at postoperative 6 hours, whereas the urine aquaporin-2 level increased later with a delayed peak at postoperative 12 hours. As a result, urine aquaporin-2 relative to the plasma arginine vasopressin level, which represents the activity of the collecting ducts and indicates predicted responses to tolvaptan, was a minimum at postoperative 6 hours. Tolvaptan administration immediately after cardiac surgery might not be recommended given the transient refractoriness to tolvaptan probably due to the stunning of kidney collecting ducts.

Congenital nephrogenic diabetes insipidus accompanied with central nephrogenic diabetes secondary to pituitary surgery -a case report

BackgroundDiabetes insipidus (DI) can be a common cause of polydipsia and polyuria. Here, we present a case of congenital nephrogenic diabetes insipidus (CNDI) accompanied with central diabetes insipidus (CDI) secondary to pituitary surgery.Case presentationA 24-year-old Chinese woman came to our hospital with the complaints of polydipsia and polyuria for 6 months. Six months ago, she was detected with pituitary apoplexy, and thereby getting pituitary surgery. However, the water deprivation test demonstrated no significant changes in urine volume and urine gravity in response to fluid depression or AVP administration. In addition, the genetic results confirmed a heterozygous mutation in arginine vasopressin receptor type 2 (AVPR2) genes.ConclusionsShe was considered with CNDI as well as acquired CDI secondary to pituitary surgery. She was given with hydrochlorothiazide (HCTZ) 25 mg twice a day as well as desmopressin (DDAVP, Minirin) 0.1 mg three times a day. There is no recurrence of polyuria or polydipsia observed for more than 6 months. It can be hard to consider AVPR2 mutation in female carriers, especially in those with subtle clinical presentation. Hence, direct detection of DNA sequencing with AVPR2 is a convenient and accurate method in CNDI diagnosis.

Abstract 3062: Drug repurposing of β-blocker propranolol and hemostatic compound desmopressin in osteosarcoma: Preclinical antitumor activity on 2D and 3D cell growth, chemotaxis and xenograft progression

Abstract Introduction. Osteosarcoma (OS) is the most common malignant primary bone tumor in children and young adults, with alarmingly elevated mortality rates, especially in developing countries. OS patients bear highly invasive and vascularized tumors, with limited response to standard of care (SoC) therapies, and are in urgent need of novel therapeutic strategies. Propranolol (PPN) is a non-selective β-adrenergic receptor (β-AR) antagonist originally used in the treatment of diverse heart diseases. On the other hand, desmopressin (dDAVP) is a hemostatic drug that acts as a selective agonist for the vasopressin type-2 receptor (AVPR2) present in blood microvessels and several tumor types. Given that β-ARs and AVPR2 signalling regulates many cellular processes involved in the initiation and progression of cancer, multiple efforts have been made to repurpose PPN and dDAVP in indications such as breast and colorectal cancer, melanoma and angiosarcoma, among others. Considering the unsatisfied clinical needs of OS, the objective of this work was to evaluate the in vitro/in vivo antitumoral activity of PPN and dDAVP on highly aggressive preclinical models of OS. Materials and methods. The human OS cell lines MG-63 and U2-OS were used for in vitro or in vivo experiments. Target expression was assessed by qPCR and immunohistochemistry. Sensitivity to PPN or dDAVP was evaluated by in vitro 72 h proliferation, 7 d clonogenic growth, 3D spheroid formation, transwell chemotaxis assays and MG-63 xenograft progression in nude mice. In addition, PPN was evaluated as monotherapy or in combination with SoC chemotherapy. Results. PPN (10-100 µM) and dDAVP (0.1-10 µM) drastically reduced clonogenic and 3D growth, migration, mitotic index and proliferation of β-AR and AVPR2-expressing OS cells (p<0.05). Synergistic antitumoral effects were observed after combining PPN with cisplatin or methotrexate, in vitro and in vivo (CI<1). In animals bearing growing OS s.c. xenografts sustained treatment during 4 weeks with PPN (10 mg/kg i.p. daily) or dDAVP (12 µg/kg i.v. three times a week) markedly abrogated tumor progression, exhibiting modulation of local tumor aggressiveness and reducing final tumor burden by 25 or 45%, respectively (p<0.01). Conclusions. Taking into account PPN and dDAVP robust antitumoral effects, β-ARs antagonization or agonist activation of AVPR2 are achievable and interesting therapeutic approaches to modulate OS tumor aggressiveness. Both agents could be proposed as coadyuvant agents for treating OS, not only in combination with chemotherapy but also administered during the perioperative setting. Citation Format: Luisina Solerno, Natasha Sobol, Rocío Rodriguez, Marina Pifano, Giselle Ripoll, Hernan Farina, Liliana Vasquez, Daniel F. Alonso, Juan Garona. Drug repurposing of β-blocker propranolol and hemostatic compound desmopressin in osteosarcoma: Preclinical antitumor activity on 2D and 3D cell growth, chemotaxis and xenograft progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3062.

Epithelial Vasopressin Type-2 Receptors Regulate Myofibroblasts by a YAP-CCN2-Dependent Mechanism in Polycystic Kidney Disease.

Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

Glucocorticoid Receptor Maintains Vasopressin‐Regulated Water Reabsorption Pathway in the Kidney Collecting Duct Cells

Aquaporin‐2 (AQP2) is a transmembrane protein expressed in the principal cells of the kidney collecting ducts, which mediate osmotic water reabsorption. AQP2 is regulated by the antidiuretic peptide hormone arginine vasopressin (AVP). When AVP binds to the vasopressin type 2 receptor (V2R) in the principal cells, it initiates a signaling network that results in 1) AQP2 trafficking to the apical plasma membrane for water transport and 2) AQP2 gene expression. In a previous study, we found that dexamethasone, a glucocorticoid receptor (GR) agonist, enhanced AVP‐induced AQP2 gene expression in the collecting duct cell model mpkCCD. Here, we showed that GR knockdown drastically decreased AVP‐induced AQP2 mRNA and protein levels, indicative of a role of GR in AVP‐induced AQP2 gene expression. We further showed that the 1,000bp region upstream to the AQP2 transcription start site was sufficient to mediate GR‐enhanced AVP‐induced promoter activity and that GR bound to the above AQP2 promoter region. RNA‐seq was used to profile the transcriptome of the GR knockdown vs. control cells. A total of 93,308 transcripts belonging to 22,025 genes were quantified. GR knockdown decreased expression of 5,266 genes and increased expression of 3,709 genes. These two gene groups respectively represent positive and negative regulators for the AVP responses. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis showed 62 pathways associated with the positive regulators and 48 pathways associated with the negative regulators. The AVP‐regulated water reabsorption pathway was among the 62 positive pathways whose gene expression was reduced upon GR knockdown. Quantitative RT‐PCR analysis showed about 60% reduction in the V2R mRNA level upon GR knockdown. Taken together, our results implicated that GR in one hand potentiates AVP‐induced AQP2 gene expression and in the other hand maintains expression of genes involved in the AVP‐regulated water reabsorption pathway in the kidney collecting duct cells.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, Taiwan (107‐2320‐B‐002‐057‐MY3).