Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting ß-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels.

Arginine-vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Desmopressin, Misoprostol, nor Carboprost Affect Platelet Aggregability Following Traumatic Brain Injury and Aspirin.

INTRODUCTION: Desmopressin (DDAVP) has been utilized clinically in patients taking aspirin (ASA) to improve drug-induced platelet dysfunction. Misoprostol and carboprost, prostaglandin analogs commonly used for postpartum hemorrhage, may also induce platelet aggregation. The aim of this study was to determine the effects of DDAVP, misoprostol, and carboprost administration on platelet aggregability following traumatic brain injury (TBI) in mice treated with ASA. METHODS: Male C57BL/6 mice were randomized into seven groups (n = 5 each): untouched, ASA only, Saline/TBI, ASA/TBI, ASA/TBI/DDAVP 0.4 µg/kg, ASA/TBI/misoprostol 1 mg/kg, and ASA/TBI/carboprost 100 µg/kg. TBI was induced via a weight drop model 4-h after ASA (50 mg/kg) gavage. Mice were given an intraperitoneal injection of DDAVP, misoprostol, or carboprost 10 minutes after TBI. In vivo testing was completed utilizing tail vein bleed. Mice were sacrificed 30-min posttreatment and blood was collected via cardiac puncture. Whole blood was analyzed via Multiplate impedance aggregometry, rotational thromboelastometry, and TEG6s. RESULTS: Mice receiving misoprostol after ASA/TBI demonstrated decreased tail vein bleeding times compared to ASA only treated mice. However, mice treated with misoprostol following ASA and TBI demonstrated decreased platelet aggregability compared to untouched mice and TBI only mice within the arachidonic acid agonist pathway. By contrast, DDAVP and carboprost did not significantly change platelet aggregability via adenosine diphosphate or arachidonic acid following ASA and TBI. However, DDAVP did decrease the platelet contribution to clot via rotational thromboelastometry. CONCLUSIONS: Reversal of medication-induced platelet inhibition has become increasingly controversial after TBI. Based on these results, DDAVP, misoprostol, nor carboprost consistently improve platelet aggregability following TBI in those also treated with ASA.

Poly(ADP-ribose) polymerase-1 affects vasopressin-mediated AQP2 expression in collecting duct cells of the kidney.

Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.

Desmopressin enhances random-pattern skin flap survival in rats: Possible role of vasopressin Type-1a and 2 receptors.

BACKGROUND: Many drugs have been explored for their role in improving skin flap survival. 1-deamino-8-D-arginine vasopressin (DDAVP or desmopressin) is a synthesized form of anti-diuretic hormone (ADH) and a selective agonist for vasopressin type-2 receptors (V2 receptors). Desmopressin has been shown to improve endothelial function, induce vasodilation, and reduce inflammation. We aimed to evaluate its efficacy in enhancing flap survival and assess the role of vasopressin receptors in this process. MATERIALS AND METHODS: We randomly assigned six male Wistar rats to each study group. Different doses of desmopressin were injected intraperitoneally to find the most effective amount (8 µg/rat). SR-49059, a selective V1a receptor antagonist, was given at 2µg/rat before providing the most effective dose of desmopressin (8µg/rat). Histopathological assessments, quantitative measurements of interleukin-1ß (IL-1ß), Tumor necrosis factor-alpha (TNF-α), and Nuclear Factor-κB (NF-κB), optical imaging, and measurement of the expression levels of V2 receptor in the rat skin tissue were performed. RESULTS: Desmopressin (8µg/rat) significantly reduced the mean percentage of necrotic area compared to the control group (19.35% vs 73.57%). Histopathological evaluations revealed a notable reduction in tissue inflammation, edema, and degeneration following administration of desmopressin (8). The expression of the V2 receptor was increased following desmopressin administration. It also led to a reduction in IL-1ß, TNF-α, and NF-κB levels. The protective effect of desmopressin on flap survival was reversed upon giving SR-49059. The optical imaging revealed enhanced blood flow in the desmopressin group compared to the control group. CONCLUSIONS: Desmopressin could be repurposed to improve flap survival. V1a and V2 receptors probably mediate this effect.

Enhancing Cushing's disease diagnosis: exploring the impact of desmopressin on ACTH gradient during BIPSS.

Introduction: The differential diagnosis between Cushing's disease (CD) and ectopic ACTH syndrome (EAS) is complex, and bilateral inferior petrosal sinus sampling (BIPSS) is considered the gold-standard test. However, BIPSS with corticotropin-releasing hormone (CRH) stimulation is rarely available. Objective: This retrospective cohort study aimed to assess the accuracy of the inferior petrosal sinus to peripheral ACTH gradient (IPS:P) before and after desmopressin stimulation for the differential diagnosis of ACTH-dependent Cushing's syndrome (CS), applying different cutoff values. Methods: A total of 50 patients (48 with CD and 2 with EAS) who underwent BIPSS were included in this study. The sensitivity and specificity of IPS:P in BIPSS before and after desmopressin stimulation were evaluated. Various cutoff values for IPS:P were examined to determine their diagnostic accuracy. Results: Using the traditional IPS:P cutoff, the sensitivity was 85.1% before stimulation, 89.6% after stimulation, and a combined sensitivity of 91.7%. Applying cutoff values of IPS:P >1.4 before and >2.8 after stimulation, the sensitivity was 87.2% and 89.6%, respectively, with a combined sensitivity of 91.7%. Receiver operating characteristic (ROC) curve analysis determined optimal cutoff values of 1.2 before stimulation and 1.57 after stimulation, resulting in a sensitivity of 93.6% and 93.8%, respectively, with a combined sensitivity of 97.9%. Specificity remained at 100% throughout all analyses. Among the 43 patients who responded positively to stimulation, 42 (97.7%) did so within the first three minutes, and all 43 (100%) did so within the first five minutes. None of the assessed clinical variables predicted the ACTH response to stimulation in BIPSS with statistical significance. Discussion: ACTH stimulation with desmopressin during BIPSS improves the accuracy of IPS:P, making it a valuable tool for investigating ACTH-dependent Cushing's syndrome. Considering the low risk of complications, we recommend the use of desmopressin stimulation during BIPSS for the differential diagnosis of ACTH-dependent CS.

cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs.

OBJECTIVE: To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. METHODS: Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 µg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. RESULTS: Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p <  0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p <  0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p <  0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. CONCLUSION: DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. OXFORD CENTRE FOR EVIDENCE-BASED MEDICINE 2011 LEVELS OF EVIDENCE: Level 5.

Application of a TEG-Platelet Mapping Algorithm to Guide Reversal of Antiplatelet Agents in Adults with Mild-to-Moderate Traumatic Brain Injury: An Observational Pilot Study.

BACKGROUND: Traumatic intracranial hemorrhages expand in one third of cases, and antiplatelet medications may exacerbate hematoma expansion. However, the reversal of an antiplatelet effect with platelet transfusion has been associated with harm. We sought to determine whether a thromboelastography platelet mapping (TEG-PM)-guided algorithm could limit platelet transfusion in patients with hemorrhagic traumatic brain injury (TBI) prescribed antiplatelet medications without a resultant clinically significant increase in hemorrhage volume, late hemostatic treatments, or delayed operative intervention. METHODS: A total of 175 consecutive patients with TBI were admitted to our university-affiliated, level I trauma center between March 2016 and December 2019: 54 preintervention patients (control) and 121 patients with TEG-PM (study). After exclusion for anticoagulant administration, availability of neuroimaging and emergent neurosurgery, 62 study patients and 37 control patients remained. Intervention consisted of administration of desmopressin (DDAVP) for nonsurgical patients with significant inhibition at the arachidonic acid or adenosine diphosphate receptor sites. For surgical patients with significant inhibition, dual therapy with DDAVP and platelet transfusion was employed. Study patients were compared with a group of historical controls, which were identified from a prospectively maintained registry and typically treated with empiric platelet transfusion. RESULTS: Median age was 75 years (interquartile range 85-67) and 77 years (interquartile range 81-65) in the TEG-PM and control patient groups, respectively. Admission hemorrhage volumes were similar (10.7 cm3 [20.1] in patients with TEG-PM vs. 14.1 cm3 [19.7] in controls; p = 0.41). There were no significant differences in admission Glasgow Coma Scale, mechanism of trauma, or baseline comorbidities. A total of 57% of controls versus 10% of patients with TEG-PM (p < 0.001) were transfused platelets; 52% of intervention patients and 0% controls were treated with DDAVP. Expansion hemorrhage volumes were not significantly different (14.0 cm3 [20.2] patients with TEG-PM versus 13.6 cm3 [23.7] controls; p = 0.93). There was no significant difference in rates of clinical deterioration, delayed neurosurgical intervention, or late platelet transfusion between groups. CONCLUSIONS: Among patients with hemorrhagic TBI prescribed preinjury antiplatelet therapy, our study suggests that the use of a TEG-PM algorithm may reduce platelet transfusions without a concurrent increase in clinically significant hematoma expansion. Further study is required to prove a causative relationship.

Physical basis for the determination of lumen shape in a simple epithelium.

The formation of a hollow lumen in a formerly solid mass of cells is a key developmental process whose dysregulation leads to diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by experiments in the mouse blastocyst. However, lumens formed in other tissues adopt irregular shapes with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen shape in all cases. Here we use live-cell imaging to study the physical mechanism of lumen formation in Madin-Darby Canine Kidney cell spheroids, a canonical cell-culture model for lumenogenesis. We find that in this system, lumen shape reflects basic geometrical considerations tied to the establishment of apico-basal polarity. A physical model incorporating both cell geometry and intraluminal pressure can account for our observations as well as cases in which pressure plays a dominant role.

Functional characterization of a loss-of-function mutant I324M of arginine vasopressin receptor 2 in X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly "rescue" I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

Vasopressin Induces Urinary Uromodulin Secretion By Activating PKA (Protein Kinase A).

[Figure: see text].

Urinary reabsorption in the rat kidney by anticholinergics.

Anticholinergics, therapeutic agents for overactive bladder, are clinically suggested to reduce urine output. We investigated whether this effect is due to bladder or kidney urine reabsorption. Various solutions were injected into the bladder of urethane-anesthetized SD rats. The absorption rate for 2 h was examined following the intravenous administration of the anticholinergics imidafenacin (IM), atropine (AT), and tolterodine (TO). The bilateral ureter was then canulated and saline was administered to obtain a diuretic state. Anticholinergics or 1-deamino-[8-D-arginine]-vasopressin (dDAVP) were intravenously administered. After the IM and dDAVP administrations, the rat kidneys were immunostained with AQP2 antibody, and intracellular cAMP was measured. The absorption rate was ~ 10% of the saline injected into the bladder and constant even when anticholinergics were administered. The renal urine among peaked 2 h after the saline administration. Each of the anticholinergics significantly suppressed the urine production in a dose-dependent manner, as did dDAVP. IM and dDAVP increased the intracellular cAMP levels and caused the AQP2 molecule to localize to the collecting duct cells' luminal side. The urinary reabsorption mechanism through the bladder epithelium was not activated by anticholinergic administration. Thus, anticholinergics suppress urine production via an increase in urine reabsorption in the kidneys' collecting duct cells via AQP2.

A metabolically stable apelin-17 analog decreases AVP-induced antidiuresis and improves hyponatremia.

Apelin and arginine-vasopressin (AVP) are conversely regulated by osmotic stimuli. We therefore hypothesized that activating the apelin receptor (apelin-R) with LIT01-196, a metabolically stable apelin-17 analog, may be beneficial for treating the Syndrome of Inappropriate Antidiuresis, in which AVP hypersecretion leads to hyponatremia. We show that LIT01-196, which behaves as a potent full agonist for the apelin-R, has an in vivo half-life of 156 minutes in the bloodstream after subcutaneous administration in control rats. In collecting ducts, LIT01-196 decreases dDAVP-induced cAMP production and apical cell surface expression of phosphorylated aquaporin 2 via AVP type 2 receptors, leading to an increase in aqueous diuresis. In a rat experimental model of AVP-induced hyponatremia, LIT01-196 subcutaneously administered blocks the antidiuretic effect of AVP and the AVP-induced increase in urinary osmolality and induces a progressive improvement of hyponatremia. Our data suggest that apelin-R activation constitutes an original approach for hyponatremia treatment.

Effect of Desmopressin on Platelet Dysfunction During Antiplatelet Therapy: A Systematic Review.

BACKGROUND AND OBJECTIVE: An increasing number of patients receive antiplatelet therapy. Patients exposed to surgery while receiving platelet inhibitors hold an increased bleeding risk. Especially in neurosurgery and neurocritical care patients, bleeding and hematoma expansion are feared complications as even minor bleedings may be hazardous. The objective of this systematic review was to investigate the effect of desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) on platelet function during antiplatelet therapy in patients undergoing non-cardiac surgery, patients who experience spontaneous or traumatic hemorrhage, healthy individuals and in animals. METHODS: Studies were identified through a systematic literature search in PubMed and EMBASE on August 19, 2019, with an update on May 2, 2020, and from reference lists of the included studies. Data on clinical and biochemical effect of DDAVP were extracted from included studies for a qualitative data synthesis. RESULTS: In total, 22 studies were included: 18 human studies and four animal studies. Overall, DDAVP improved bleeding time and increased platelet aggregation in patients undergoing non-cardiac surgery, patients suffering intracerebral or subarachnoid hemorrhage while receiving antiplatelet therapy as well as in healthy individuals and animals exposed to antiplatelet therapy. Observational data indicate that DDAVP may mitigate hematoma expansion in patients with intracerebral hemorrhage or traumatic brain injury. CONCLUSIONS: The present data hold biochemical evidence that DDAVP improves platelet function during antiplatelet therapy in humans and animals. The need for randomized trials is evident in order to evaluate the potential clinical effect of DDAVP in management of patients with spontaneous or traumatic hemorrhage, or undergoing neurosurgery, while receiving antiplatelet therapy.

The aptamer BT200 effectively inhibits von Willebrand factor (VWF) dependent platelet function after stimulated VWF release by desmopressin or endotoxin.

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state.

Differential Osteoprotegerin Kinetics after Stimulation with Desmopressin and Lipopolysaccharides In Vivo.

Osteoprotegerin (OPG) regulates bone metabolism by reducing the activation of osteoclasts, but may also be involved in blood vessel calcification and atherosclerosis. Within endothelial cells OPG is stored in Weibel-Palade bodies (WPBs). Blood kinetics of OPG are essentially unknown. We aimed to assess these using two distinct in vivo models; one after stimulation with desmopressin (DDAVP) and another after stimulation with lipopolysaccharide (LPS). Both clinical trials were conducted at the Department of Clinical Pharmacology at the Medical University of Vienna, Austria. Participants received desmopressin (0.3 µg/kg), LPS (2 ng/kg), or placebo (sodium chloride 0.9%) with subsequent blood sampling at time points up to 24 hours after administration. The primary objective of this study was to investigate the plasma kinetics of OPG after stimulation with desmopressin and LPS. Secondary analyses included the release of other WPB contents including von Willebrand factor (vWF). This analysis included 31 healthy volunteers (n = 16 for desmopressin and placebo, n = 15 for LPS). Infusion of desmopressin did not increase OPG concentrations compared with placebo, while LPS infusion significantly increased OPG levels, both compared with desmopressin (p < 0.0001) and to placebo (p = 0.004), with a maximum of ∼twofold increase in OPG levels ∼6 hours after infusion. von Willebrand factor levels increased after both desmopressin and LPS infusion (p < 0.0001), with a maximum of ∼threefold increase 2 hours after desmopressin and a maximum of ∼twofold increase 6 hours after LPS administration. In conclusion, we report that, in contrast to vWF, OPG is not released upon stimulation with desmopressin, but increases significantly during experimental endotoxemia.

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

A prospective randomized trial of desmopressin in canine mammary carcinoma.

Metastatic disease represents a serious and often fatal development in patients with solid tumours, including women with breast cancer and dogs with mammary tumours. Therefore, preventing and treating metastatic disease has remained a priority in cancer research. Desmopressin, a synthetic derivative of vasopressin, traditionally used to treat patients with bleeding disorders, has been proposed as a potential anti-metastatic agent due to its effect on haemostasis as well as multiple other anti-proliferative and anti-angiogenic mechanisms. The purpose of this study was to retest desmopressin in dogs with mammary carcinomas. A prospective randomized study was performed. Twenty-four dogs with mammary carcinomas were enrolled; 12 dogs received perioperative desmopressin and 12 received placebo. All dogs underwent standard pre-surgical staging followed by complete resection of all tumours. Intact dogs were spayed. All tumours were graded and classified according to the published guidelines. Follow-up was performed every 4 months the first year and every 6 months thereafter. Necropsies were requested on all dogs. There was no difference in time to primary metastasis or survival between desmopressin treated dogs and the placebo arm (P = .43 and .73, respectively). The distribution of negative prognostic factors, including tumour grade, stage, and high vs low bioscore (refined flexible bioscoring) category between arms was not statistically different, even though more dogs in the placebo arm had grade 3 tumours and high bioscores. Based on the results of this study, perioperative desmopressin does not prevent metastasis in dogs with mammary carcinomas.

Investigating hormone-induced changes in affective state using the affective bias test in male and female rats.

Recent clinical and pre-clinical research suggests that affective biases may play an important role in the development and perpetuation of mood disorders. Studies in animals have also revealed that similar neuropsychological processes can be measured in non-human species using behavioural assays designed to measure biases in learning and memory or decision-making. Given the proposed links between hormones and mood, we used the affective bias test to investigate the effects of different hormone treatments in both male and female rats. Animals were pre-treated with acute doses of hormone or vehicle control prior to learning each of two independent substrate-reward associations. During a subsequent choice test, positive or negative biases were observed by animal's preference towards or away from the substrate learnt during drug treatment respectively. In both sexes, oestradiol and the oestrogen-like compound bisphenol A induced positive biases, whilst blockade of oestrogen hormones with formestane induced a negative bias. Progesterone induced a negative bias in both sexes, but testosterone only induced a negative bias in males. Blocking testosterone with flutamide induced a positive bias in both sexes at the higher dose (10 mg/kg). The oxytocin analogue, carbetocin induced positive biases in both sexes but the vasopressin analogue, desmopressin, induced a positive bias in male rats only. These results provide evidence that modulating levels of hormones using exogenous treatments can induce affective biases in rats. They also suggest that hormone-induced affective biases influence cognitive and emotional behaviour and could have longer-term effects in some mood disorders.

Development and therapeutic potential of vasopressin synthetic analog [V4Q5]dDAVP as a novel anticancer agent.

Since its discovery, arginine vasopressin (AVP) was subjected to several modifications with the aim of obtaining novel derivatives with increased potency and selectivity for biomedical use. Desmopressin (dDAVP) is a first generation synthetic analog of AVP with hemostatic and antimetastatic activity. dDAVP acts as a selective agonist of the arginine vasopressin type 2 receptor (AVPR2) present in microvascular endothelium and cancer cells. Considering its selective effects on AVPR2-expressing malignant and vascular tissue, and interesting antitumor profile, dDAVP was used as a lead compound for the development of novel peptide analogs with enhanced anticancer efficacy. After conducting different structure-activity relationship studies to determine key aminoacidic positions for its antitumor activity against AVPR2-expressing malignant cells, dDAVP was rationally modified and a wide panel of synthetic analogs with different sequence and structural modifications was assessed. As a result of this structure-based drug derivatization novel AVP analog [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-d-arginine vasopressin) was selected as the most active candidate and further developed. [V4Q5]dDAVP was evaluated in highly aggressive and metastatic cancer preclinical models deploying enhanced cytostatic, antimetastatic and angiostatic effects in comparison to parental peptide dDAVP. In addition, novel compound demonstrated good tolerability as evaluated in several toxicological studies, and cooperative therapeutic effects after combination with standard-of-care chemotherapy. In summary, due to its ability to inhibit growth and tumor-associated angiogenesis, as well as impairing progression of metastatic disease, AVP analogs such as novel [V4Q5]dDAVP are promising compounds for further development as coadjuvant agents for the management of advance or recurrent cancers.