Vasopressin Stimulation Research Articles

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.

THU037 Diagnostic Accuracy Of Bilateral Inferior Petrosal Sinus Sampling In Adrenocorticotropin-dependent Cushing’s Syndrome, And The Utility Of Maximum-stimulated Petrosal Sinus ACTH To Baseline Petrosal Sinus ACTH Ratio

Abstract Disclosure: A.N. Maldar: None. P.H. Chauhan: None. A. Kulkarni: None. N.F. Shah: None. B.K. Misra: None. M. Chadha: None. Methods: The diagnosis of Cushing’s Syndrome (CS) was made as per the prevalent endocrine society guidelines. CS patients with serum ACTH>15 pg/mL were considered as ACTH-dependent CS. Baseline ACTH (both inferior petrosal sinuses and peripheral) values were obtained, and since 2014, stimulated ACTH values (5 and 10 minutes post one unit iv vasopressin) were obtained as standard protocol. Prolactin corrected ACTH ratios were additionally being calculated since 2016. The highest central to peripheral ACTH gradient >2 in baseline samples (bC:P), >3 in samples after vasopressin stimulation (sC:P), and >0.8 in PRL-corrected ratios (pC:P) was used as reference value indicative of Cushing’s Disease (CD). An inter-petrosal gradient >1.4 was considered to be suggestive of an adenoma located on the side of the petrosal sinus with the higher ACTH concentration. Results: Of the 51 patients with ACTH-dependent CS who underwent BIPSS, four patients with incomplete data, one patient with unilateral petrosal sinus catheterization, one patient with Cortisol Resistance Syndrome were excluded from the analysis. Mean age of our cohort was 37.5 years, with 77.8% females. 39 patients had CD (positive histopathology-immunohistochemistry in 34, and remission post-surgery in additional five with hyperplasia), while six had ectopic CS (one each of bronchial carcinoid, lung NET, thymic NET, pancreatic NET, and two with undiagnosed ectopic source). bC:P>2 (N=45) demonstrated 92.3% sensitivity, 83.3% specificity, 97.3% PPV and 62.5% NPV, while sC:P>3 (N=31) demonstrated 96.3% sensitivity, 100% specificity, 100% PPV and 80% NPV to diagnose CD. pC:P>0.8 (N=20) improved the sensitivity and NPV to 100%, with a 75% specificity and 94.1% PPV. On ROC analysis, cut-point ratio of 2.55 for bC:P value gave a sensitivity of 89.7% and specificity of 100% (AUC=0.964); sC:P ratio of 2.95 had 96.3% sensitivity and 100% specificity (AUC=0.991); 0.93 for pC:P ratio provided a sensitivity of 93.8% and specificity of 100% for diagnosis of CD (AUC=0.984). Maximum-stimulated petrosal sinus ACTH to baseline petrosal sinus ACTH ratio of <1.11 provided a 100% sensitivity, while a ratio >1.75 provided 100% specificity towards diagnosis of CD. 71.4% (10/14) patients with left sided adenoma, and 85% (17/20) patients with right sided adenoma were correctly lateralised on BIPSS. In patients whom adenoma was detected on pituitary MRI, 66.7% (6/9) were correctly lateralised to the left side, and 94.1% (16/17) to the right side. Conclusion: BIPSS provides high sensitivity and specificity to diagnose CD. Vasopressin stimulation improves the sensitivity and specificity, while prolactin correction improves the sensitivity of BIPSS, as compared to baseline ratios. Maximum-stimulated petrosal sinus ACTH to baseline petrosal sinus ACTH ratio can be additionally used, especially in CD patients suspected of false negative localization. Presentation: Thursday, June 15, 2023

The fructose survival hypothesis for obesity.

The fructose survival hypothesis proposes that obesity and metabolic disorders may have developed from over-stimulation of an evolutionary-based biologic response (survival switch) that aims to protect animals in advance of crisis. The response is characterized by hunger, thirst, foraging, weight gain, fat accumulation, insulin resistance, systemic inflammation and increased blood pressure. The process is initiated by the ingestion of fructose or by stimulating endogenous fructose production via the polyol pathway. Unlike other nutrients, fructose reduces the active energy (adenosine triphosphate) in the cell, while blocking its regeneration from fat stores. This is mediated by intracellular uric acid, mitochondrial oxidative stress, the inhibition of AMP kinase and stimulation of vasopressin. Mitochondrial oxidative phosphorylation is suppressed, and glycolysis stimulated. While this response is aimed to be modest and short-lived, the response in humans is exaggerated due to gain of 'thrifty genes' coupled with a western diet rich in foods that contain or generate fructose. We propose excessive fructose metabolism not only explains obesity but the epidemics of diabetes, hypertension, non-alcoholic fatty liver disease, obesity-associated cancers, vascular and Alzheimer's dementia, and even ageing. Moreover, the hypothesis unites current hypotheses on obesity. Reducing activation and/or blocking this pathway and stimulating mitochondrial regeneration may benefit health-span. This article is part of a discussion meeting issue 'Causes of obesity: theories, conjectures and evidence (Part I)'.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

ODP302 Cushing's Disease: A Challenging Diagnosis in Patients with Underlying Co-Morbidities

Abstract Introduction Cushing's disease (CD) is a rare endocrine disorder with significant morbidity and mortality. This case is a complicated ACTH (adrenocorticotropic hormone)-dependent hypercortisolism with a negative pituitary MRI inthe setting of underlying liver cirrhosis and chronic kidney disease. Hypercortisolism work up was challenging due to liver and kidney disease. Case Presentation A 42-year-old woman with a history of liver cirrhosis(MELDscore: 19) and stage 3b chronic kidney disease (CKD) presented with nausea, vomiting and abdominal pain. She reported proximal muscle weakness. On examination, she had central adiposity with large purple abdominal striae and proximal muscle wasting. Medical history was pertinent for uncontrolled hypertension and type 2 diabetes mellitus. Due to concern for Cushing's syndrome, an overnight 1 mg dexamethasone suppression test (DST) was performed. She had an inappropriately elevated morning cortisol of 10.1 mcg/dL (n < 1.8 mcg/dL), but suboptimal dexamethasone level of 104 ng/dL (140-295 ng/dL), suggesting incomplete absorption secondary to emesis. A repeat DST was not performed given concerns for reduced dexamethasone clearance with her underlying renal and liver disease. Her 24-hour urine free cortisol (UFC) was elevated at 55 ug/24 hr (6-42 ug/24 hr). A repeat 24-hour UFC was again elevated at 265 ug/24 hr (6-42 ug/24 hr). Three late night salivary cortisol levels were also elevated at 0.231 ug/dL, 0.429 ug/dL and 0.19 ug/dL (n <0. 01-0. 09 ug/dL). Diagnosis of Cushing's syndrome was confirmed. ACTH level obtained was detectable at 70.1 pg/mL (7.2-63.3 pg/mL), suggestive of ACTH-dependent Cushing's syndrome. Pituitary MRI without contrast was obtained due to CKD. MRI reported no pituitary abnormalities. High dose DST was not performed due to concerns for aforementioned reduced dexamethasone clearance and due to lack of availability of CRH (corticotropin-releasing hormone), CRH stimulation testing was not done. She underwent inferior petrosal sinus sampling (IPSS) which showed central to peripheral ACTH ratio of 2.11 at baseline and ratios of 3.53, 3.45 and 3.32 at 5 minutes, 10 minutes and 15 minutes respectively after vasopressin stimulation, solidifying the diagnosis of Cushing's disease. Patient is now scheduled for an exploration of the sella via endoscopic endonasal approach. Conclusion This case highlights the limitations of biochemical tests and imaging modalities in the workup of hypercortisolism, notably in patients with underlying liver and kidney disease. The ordering physician should be mindful of the caveats associated with each biochemical testing. ACTH-dependent Cushing's syndrome can be difficult to diagnose properly in the setting of a negative pituitary MRI. IPSS, although an expensive and invasive test, has the best diagnostic accuracy. This case further demonstrates the importance of performing an IPSS to diagnose CD and distinguish from an ectopic source when imaging is non-diagnostic. Presentation: No date and time listed

AQP2 trafficking in health and diseases: an updated overview.

Renal collecting duct principal cells play a key role in controlling body water balance. Principal cells express the water channels AQP2, AQP3, and AQP4 that mediate renal water reabsorption. AQP3 and AQP4 are expressed at the basolateral membrane constitutively. Conversely, AQP2 is localized in intracellular vesicles and translocates to the plasma membrane under vasopressin action. Stimulation with vasopressin activates the cAMP/PKA signal transduction pathway that induces the redistribution of AQP2 from an intracellular pool to the apical plasma membrane. AQP2 trafficking and function depend on multiple post-translational modifications. Moreover, several proteins control different steps activated by the vasopressin stimulation that triggers the redistribution of the AQP2 vesicles. A-kinase anchoring proteins (AKAPs) together with phosphodiesterases and adenylate cyclases play crucial roles in modulating local changes of cAMP. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE), cytoskeletal proteins, and the small GTPases of the Rho family regulate the fusion and the endocytotic retrieval of AQP2 vesicles. Abnormal vasopressin signaling and altered AQP2 expression or trafficking can lead to disorders characterized by deregulated mechanisms controlling water homeostasis. This review provides updated data on the molecular signals regulating vasopressin-induced AQP2 trafficking in health and disease.

Current Hydration Habits: The Disregarded Factor for the Development of Renal and Cardiometabolic Diseases.

Improper hydration habits are commonly disregarded as a risk factor for the development of chronic diseases. Consuming an intake of water below recommendations (underhydration) in addition to the substitution of sugar-sweetened beverages (SSB) for water are habits deeply ingrained in several countries. This behavior is due to voluntary and involuntary dehydration; and because young children are exposed to SSB, the preference for a sweet taste is profoundly implanted in the brain. Underhydration and SSB intake lead to mild hyperosmolarity, which stimulates biologic processes, such as the stimulation of vasopressin and the polyol-fructose pathway, which restore osmolarity to normal but at the expense of the continued activation of these biological systems. Unfortunately, chronic activation of the vasopressin and polyol-fructose pathways has been shown to mediate many diseases, such as obesity, diabetes, metabolic syndrome, chronic kidney disease, and cardiovascular disease. It is therefore urgent that we encourage educational and promotional campaigns that promote the evaluation of personal hydration status, a greater intake of potable water, and a reduction or complete halting of the drinking of SSB.

Functional Rescue of a Nephrogenic Diabetes Insipidus Causing Mutation in the V2 Vasopressin Receptor by Specific Antagonist and Agonist Pharmacochaperones.

The urine concentrating function of the kidney is essential to maintain the water homeostasis of the human body. It is mainly regulated by the arginine-vasopressin (AVP), which targets the type 2 vasopressin receptor (V2R) in the kidney. The inability of V2R to respond to AVP stimulation leads to decreased urine concentration and congenital nephrogenic diabetes insipidus (NDI). NDI is characterized by polyuria, polydipsia, and hyposthenuria. In this study, we identified a point mutation (S127F) in the AVPR2 gene of an NDI patient, and we characterized the impaired function of the V2R mutant in HEK293 cells. Based on our data, the S127F-V2R mutant is almost exclusively located intracellularly in the endoplasmic reticulum (ER), and very few receptors were detected at the cell surface, where the receptor can bind to AVP. The overexpressed S127F-V2R mutant receptor has negligible cAMP generation capability compared to the wild-type receptor in response to AVP stimulation. Since certain misfolded mutant proteins, that are retained in the ER, can be rescued by pharmacological chaperones, we examined the potential rescue effects of two pharmacochaperones on the S127F-V2R. We found that pretreatment with both tolvaptan (an established V2R inverse agonist) and MCF14 compound (a cell-permeable high-affinity agonist for the V2R) were capable of partially restoring the cAMP generating function of the receptor in response to vasopressin stimulation. According to our data, both cell permeant agonists and antagonists can function as pharmacochaperones, and serve as the starting compounds to develop medicines for patients carrying the S127F mutation.

Clinical, Genetic and Functional Characterization of a Novel AVPR2 Missense Mutation in a Woman with X-Linked Recessive Nephrogenic Diabetes Insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by renal unresponsiveness to the hormone vasopressin, leading to excretion of large volumes of diluted urine. Mutations in the arginine vasopressin receptor-2 (AVPR2) gene cause congenital NDI and have an X-linked recessive inheritance. The disorder affects almost exclusively male family members, but female carriers occasionally present partial phenotypes due to skewed inactivation of the X-chromosome. Here, we report a rare case of a woman affected with X-linked recessive NDI, presenting an average urinary output of 12 L/day. Clinical and biochemical studies showed incomplete responses to water deprivation and vasopressin stimulation tests. Genetic analyses revealed a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro) in the AVPR2 gene. Using a combination of in-silico protein modeling with human cellular models and molecular phenotyping, we provide functional evidence for phenotypic effects. The mutation destabilizes the helical structure of the AVPR2 transmembrane domains and disrupts its plasma membrane localization and downstream intracellular signaling pathways upon activation with its agonist vasopressin. These defects lead to deficient aquaporin 2 (AQP2) membrane translocation, explaining the inability to concentrate urine in this patient.

Bayesian Identification of Transcription Factors that Regulate Aqp2 Transcription

Background Renal collecting duct and connecting tubule cells selectively express the water channel aquaporin-2 (AQP2) and Aqp2 gene transcription is strongly regulated by vasopressin. However, the transcription factors (TFs) responsible for regulation of expression of AQP2 remain largely unknown. Here, we used Bayesian data integration techniques to identify these TFs. Method The general strategy is to use Bayes’ Rule to integrate several -omic datasets to stratify a curated list of 1344 TFs present in the mouse genome with regard to probability of regulating Aqp2 gene transcription. First, existing proteomic and transcriptomic data were used to prioritize TFs according to expression level in mpkCCD and native IMCD cells. Second, we used our existing ATAC-Seq, histone H3K27-acetylation ChIP-Seq, and RNA-polymerase II ChIP-Seq data to identify enhancer regions in the CTCF loop surrounding the Aqp2 gene. The sequences within these enhancers were analyzed to identify recognized TF binding motifs within them; and these motifs were matched to TFs on the Bayesian list to identify the TFs most likely to bind Aqp2 regulatory regions. Beyond this, we use additional -omic datasets to prioritize TFs that are regulated by vasopressin. Finally, we carried out new RNA-Seq experiments mapping the time course of vasopressin induced changes in the transcriptome of mouse mpkCCD cells to further prioritize TFs that change in tandem with AQP2. Results The analysis identified 21 TFs out of 1344 in the mouse genome that are most likely to be involved in regulation of Aqp2 gene transcription. These TFs included nine that have been proposed in prior studies to play a role in Aqp2 regulation, viz. Cebpb, Elf1, Elf3, Ets1, Hes1, Jun, Junb, Nfkb1, and Sp1. The remaining 12 represent new candidates for future studies (Atf1, Cebpg, Fosl2, Irf3, Klf5, Klf6, Mef2d, Nfib, Nfyb, Nfyc, Nr2f6, Stat3). Conspicuously absent is CREB, i.e. Creb1, which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription. Instead, another CREB-like TF, Atf1, with a pKID PKA-phosphorylation domain, ranked second among all transcription factors, representing an apt target for future studies. The RNA-Seq time course experiments in mpkCCD cells showed a rapid increase in Aqp2 mRNA, within 3 hour of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, a so-called pioneer TF, which we have shown by ChIP-seq studies to bind to an enhancer 938 bp downstream from the Aqp2 gene. Conclusions The Bayesian analysis has identified the TFs most likely to bind to these regions and likely to be regulated by vasopressin stimulation, providing a roadmap for future studies to understand regulation of Aqp2 gene expression.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Valine-279 Deletion-Mutation on Arginine Vasopressin Receptor 2 Causes Obstruction in G-Protein Binding Site: A Clinical Nephrogenic Diabetes Insipidus Case and Its Sub-Molecular Pathogenic Analysis.

Congenital nephrogenic diabetes insipidus (CNDI) is a genetic disorder caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 genes, rendering collecting duct cells insensitive to the peptide hormone arginine vasopressin stimulation for water reabsorption. This study reports a first identified AVPR2 mutation in Taiwan and demonstrates our effort to understand the pathogenesis caused by applying computational structural analysis tools. The CNDI condition of an 8-month-old male patient was confirmed according to symptoms, family history, and DNA sequence analysis. The patient was identified to have a valine 279 deletion–mutation in the AVPR2 gene. Cellular experiments using mutant protein transfected cells revealed that mutated AVPR2 is expressed successfully in cells and localized on cell surfaces. We further analyzed the pathogenesis of the mutation at sub-molecular levels via long-term molecular dynamics (MD) simulations and structural analysis. The MD simulations showed while the structure of the extracellular ligand-binding domain remains unchanged, the mutation alters the direction of dynamic motion of AVPR2 transmembrane helix 6 toward the center of the G-protein binding site, obstructing the binding of G-protein, thus likely disabling downstream signaling. This study demonstrated that the computational approaches can be powerful tools for obtaining valuable information on the pathogenesis induced by mutations in G-protein-coupled receptors. These methods can also be helpful in providing clues on potential therapeutic strategies for CNDI.

UNCOMMON AMONGST COMMON: ANGIOTENSIN II-INDUCED PERIPHERAL ISCHEMIA IN REFRACTORY SEPTIC SHOCK

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Angiotensin (AT) II, a naturally occurring hormone, modulates blood pressure through direct vasoconstriction of the circulatory system and through stimulation of various catecholamines and vasopressin. Derived from the hydrolysis of AT I via the AT-converting enzyme (ACE) in the pulmonary & renal endothelium, AT II has been studied for cases of vasodilatory shock since the 1930’. Most recently, the ATHOS-3 trial demonstrated AT II’s utility as a rescue vasopressor agent, highlighting its catecholamine-sparing effects. CASE PRESENTATION: 34-year-old African American male presented with 3-day history of dyspnea, productive cough and an episode of hemoptysis. Upon presentation, he was febrile, tachycardic, hypotensive and hypoxic prompting endotracheal intubation and vasopressor support. Laboratory studies were significant for leukocytosis with bandemia, lactic acidosis, acute kidney injury, transaminitis & positive for influenza A. Despite aggressive critical care management with maximum ventilator requirements, broad-spectrum antibiotics, renal replacement therapy & proning, his overall condition continued to worsen. AT II was initiated on Day 2 of ICU admission for refractory hypotension secondary to septic shock. Approximately 48-72 hours after the initiation of AT II, he developed skin discoloration and blistering in all extremities secondary to both arterial and venous thromboemboli despite prophylaxis. The lesions ultimately progressed leading to gangrenous necrosis extending proximally to the torso. Unfortunately, on day 7 of ICU admission, patient suffered a cardiac arrest and expired despite ACLS efforts. DISCUSSION: In our patient, we exhibited similar results to those described in the ATHOS-3 trials. Administration of the AT II resulted in an overall improvement in the patient’s mean arterial pressure, systemic vascular resistance and reduced norepinephrine requirements. We also observed a concomitant improvement in serum lactate, indicating improvement in overall organ perfusion. There are no absolute contraindications to AT II administration, but ATHOS-3 did report an increased incidence of both venous and arterial thromboembolic events in patients who received AT II. Although AT II has been FDA approved for the management of refractory septic shock, more studies are necessary to define those populations most at risk of the adverse effects. CONCLUSIONS: AT II is an important vasopressor agent used in refractory shock; however, more studies are needed to understand the adverse effects in populations most at risk. Reference #1: National Center for Biotechnology Information. PubChem Database. Angiotensin II, CID=172198, https://pubchem.ncbi.nlm.nih.gov/compound/Angiotensin-II (accessed on Mar. 26, 2020) DISCLOSURES: No relevant relationships by Krupa Gandhi, source=Web Response No relevant relationships by Mohit Mody, source=Web Response

Intracellular location of aquaporin-2 serine 269 phosphorylation and dephosphorylation in kidney collecting duct cells.

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption by the kidney collecting ducts. Under control conditions, most AQP2 resides in the recycling endosomes of principal cells, where it answers to vasopressin with trafficking to the apical plasma membrane to increase water reabsorption. Upon vasopressin withdrawal, apical AQP2 retreats to the early endosomes before joining the recycling endosomes for the next vasopressin stimulation. Prior studies have demonstrated a role of AQP2 S269 phosphorylation in reducing AQP2 endocytosis, thereby prolonging apical AQP2 retention. Here, we studied where in the cells S269 was phosphorylated and dephosphorylated in response to vasopressin versus withdrawal. In mpkCCD collecting cells, vacuolar protein sorting 35 knockdown slowed vasopressin-induced apical AQP2 trafficking, resulting in AQP2 accumulation in the recycling endosomes where S269 was phosphorylated. Rab7 knockdown, which impaired AQP2 trafficking from the early to recycling endosomes, reduced vasopressin-induced S269 phosphorylation. Rab5 knockdown, which impaired AQP2 endocytosis, did not affect vasopressin-induced S269 phosphorylation. Upon vasopressin withdrawal, S269 was not dephosphorylated in Rab5 knockdown cells. In contrast, S269 dephosphorylation upon vasopressin withdrawal was completed in Rab7 or vacuolar protein sorting 35 knockdown cells. We conclude that S269 is dephosphorylated during Rab5-mediated AQP2 endocytosis before AQP2 joins the recycling endosomes upon vasopressin withdrawal. While in the recycling endosomes, AQP2 can be phosphorylated at S269 in response to vasopressin before apical trafficking.

P0013MAP KINASES-REGULATED PROTEINS IN SIGNALING PATHWAYS OF VASOPRESSIN IN KIDNEY COLLECTING DUCT

Abstract Background and Aims Activation of G protein-coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R. However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2 expression and phosphorylation, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R. Tripartite motif-containing protein 28 (TRIM28) was identified as a substrate of ERK1 as well as a vasopressin-responsive protein via bioinformatic tools. We further evaluated whether TRIM28 plays a role in vasopressin-mediated regulation of AQP2 in the kidney CD. Method To identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on high-throughput profiles of transcriptome and proteome (http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); and 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate protein (TRIM28) was examined by immunohistochemistry. The role of TRIM28 in vasopressin-mediated AQP2 regulation was examined by quantitative real-time PCR (qRT-PCR) and semiquantitative immunoblotting after RNA interference of TRIM28 in mpkCCDc11 cells. Results Immunoblotting of mpkCCDc11 cells revealed that both p-ERK1/2 and pS261-AQP2 expression was decreased in response to dDAVP (10-9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase-substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed TRIM28, as the substrate of ERK1, has the only one phosphorylation site which was down-regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin-protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP-induced AQP2 mRNA and protein up-regulation was significantly attenuated in mpkCCDc11 cells with siRNA-mediated knockdown of TRIM28. Conclusion We identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.

SAT-LB122 Characterization and Functional Rescue of a Nephrogenic Diabetes Insipidus Causing S127F Substitution in V2 Vasopressin Receptor

Laura Szalai1, András Sziráki1, László Sándor Erdélyi1, András Balla1,2, László Hunyady1,2 1Department of Physiology, Semmelweis University, and 2MTA-SE Laboratory of Molecular Physiology, Budapest, HungaryThe concentrating function of the kidney is important to maintain the water homeostasis of the body. It is regulated by the arginine-vasopressin system through the type 2 vasopressin receptor (V2R). Loss-of-function mutations of V2R in kidney can lead to nephrogenic diabetes insipidus (NDI) which results several symptoms such as polyuria, polydipsia, and hyposthenuria.In this study, we functionally characterized and investigated the potential rescue of a missense mutation (S127F) of the V2R. We monitored the cellular localization of the S127F mutant V2 receptor using HA-tagged receptors in confocal microscopy experiments. The S127F V2 receptor was detected only in the endoplasmic reticulum but not in the plasma membrane. Flow cytometry measurements revealed that only limited amount mutant receptor can be found on the cell surface compared to the wild type V2R. We also determined the cAMP signaling capability of the mutant receptor with BRET measurements. The S127F receptor was not able to increase the intracellular cAMP levels in response to vasopressin stimulation. Certain ER retention mutations can be rescued by pharmacological chaperones, which cause misfolded mutant receptors to present in the plasma membrane. We examined the effect of tolvaptan (a V2R antagonist) on the S127F V2 receptor. HEK293 cells were transiently transfected with the plasmid of the mutant receptor and after one day the cells were incubated for 18 hours with tolvaptan. After the pretreatment, the cells were exposed to vasopressin, and we were able to detect significant cAMP signal generation of the mutant receptor. We also checked whether the result after tolvaptan pretreatment was due to restored plasma membrane location of the receptor. We were able to demonstrate significant increase of the mutant receptors in the plasma membrane using flow cytometry. We also investigated the effect of MCF14 compound (a cell permeable high-affinity agonist for the V2R) on the mutant receptor and we found that the MCF14 was also capable to restore the cAMP signaling function of the receptor.This work was supported by the National Research, Development and Innovation Fund (NKFI K116954 and NVKP_16-1-2016-0039).

MAP kinases‐Regulated Proteins in Downstream Signaling Pathways of Vasopressin V2 Receptor in Kidney Collecting Duct

BackgroundActivation of G protein‐coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R, which affects AQP2 phosphorylation (Ser261). However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R.MethodsTo identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on highthroughput profiles of transcriptome and proteome ( http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate proteins was examined by immunohistochemistry. Substrate protein‐mediated AQP2 regulation was examined quantitative real‐time PCR (qRT‐PCR) and semiquantitative immunoblotting.ResultsImmunoblotting of mpkCCDc11 cells revealed that both p‐ERK1/2 and pS261‐AQP2 expression was decreased in response to dDAVP (10−9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase‐substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed that tripartite motif‐containing 28 (TRIM28), as the substrate of ERK1, has the only one phosphorylation site which was down‐regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin‐protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP‐induced AQP2 mRNA and protein up‐regulation was significantly attenuated in mpkCCDc11 cells with siRNA‐mediated knockdown of TRIM28.ConclusionsWe identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.Support or Funding InformationThis work was supported by the National Research Foundation of Korea funded by the Ministry of Science, Information and Communication Technology, Korea (2019R1A2C1007753).

Bilateral Inferior Petrosal Sinus Sampling in Corticotropin-Dependent Cushing's Syndrome: A Single Center Experience from Western India.

Objective:Efficacy of bilateral inferior petrosal sinus sampling (BIPSS) in corticotropin-dependent Cushing's syndrome (CS) for localization and lateralization of excess adrenocorticotropic hormone (ACTH) source, as compared to high-dose dexamethasone suppression test (HDDST) and magnetic resonance imaging (MRI) pituitary, respectively.Methodology:Thirteen patients with clinically and biochemically confirmed CS underwent HDDST, MRI pituitary, and BIPSS by an experienced team of intervention neurologist, neurosurgeon, and endocrinologist using percutaneous femoral vein approach.Results:Of 13 patients (11 adults and two children) who underwent BIPSS, raised central to peripheral ACTH ratio was achieved in 12 cases, remaining one case being ectopic ACTH secretion (EAS). However, inter IPS gradient >1.4 was achieved in 11 (91.6%) of 12 Cushing's disease (CD) cases before vasopressin stimulation; and in 9 (75%) of 12 CD cases after vasopressin stimulation (P-value 0.583). HDDST suppression of more than 50% was present in only ten cases with CD, falsely negating CD in two cases (16.6%), sensitivity 83.3% and specificity 100%. MRI sella demonstrated pituitary microadenoma in 12 cases and macroadenoma in one case. Lateralization by BIPSS and MRI was concordant in 7 (58.3%) out of 12 cases with CD, with rate of remission after transsphenoidal surgery being higher in patients with concordant lateralization by BIPSS and MRI.Conclusions:BIPSS is an important investigation to distinguish CD and EAS. BIPSS was superior to HDDST for confirming the source of excess ACTH. Our findings favor the use of BIPSS for localization and pituitary MRI for lateralization of microadenoma.

Osmotic stimulation of vasopressin acutely impairs glucose regulation: a counterbalanced, crossover trial

Epidemiological studies in humans show increased concentrations of copeptin, a surrogate marker of arginine vasopressin (AVP), to be associated with increased risk for type 2 diabetes. To examine the acute and independent effect of osmotically stimulated AVP, measured via the surrogate marker copeptin, on glucose regulation in healthy adults. Sixty subjects (30 females) participated in this crossover design study. On 2 trial days, separated by ≥7 d (males) or 1 menstrual cycle (females), subjects were infused for 120 min with either 0.9% NaCl [isotonic (ISO)] or 3.0% NaCl [hypertonic (HYPER)]. Postinfusion, a 240-min oral-glucose-tolerance test (OGTT; 75 g) was administered. During HYPER, plasma osmolality and copeptin increased (P<0.05) and remained elevated during the entire 6-h protocol, whereas renin-angiotensin-aldosterone system hormones were within the lower normal physiological range at the beginning of the protocol and declined following infusion. Fasting plasma glucose did not differ between trials (P>0.05) at baseline and during the 120 min of infusion. During the OGTT the incremental AUC for glucose from postinfusion baseline (positive integer) was greater during HYPER (401.5±190.5 mmol/L·min) compared with the ISO trial (354.0±205.8 mmol/L·min; P<0.05). The positive integer of the AUC for insulin during OGTT did not differ between trials (HYPER 55,850±36,488 pmol/L·min compared with ISO 57,205±31,119 pmol/L·min). Baseline values of serum glucagon were not different between the 2 trials; however, the AUC of glucagon during the OGTT was also significantly greater in HYPER (19,303±3939 ng/L·min) compared with the ISO trial (18,600±3755 ng/L·min; P<0.05). The present data indicate that acute osmotic stimulation of copeptin induced greater hyperglycemic responses during the oral glucose challenge, possibly due to greater glucagon concentrations.This study was registered at clinicaltrials.gov as NCT02761434.

Gain-of-function mutations of the V2 vasopressin receptor in nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a cell-based assay to assess constitutive water reabsorption.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors.