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Ca2+/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in the regulation of cellular processes in a variety of tissues including brain, heart, liver, and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phosphoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs targeting the CAMK2D catalytic domain to create multiple CAMK2D KO cell lines. AQP2 protein abundance was lower in the CAMK2D KO cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D KO clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro phosphorylation studies using recombinant CAMK2D. Thirty five of the significantly downregulated phosphorylation sites in CAMK2D KO cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteómica , Acuaporina 2/genética , Acuaporina 2/metabolismo , Cromatografía Liquida , Sistemas CRISPR-Cas , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Fosforilación , Espectrometría de Masas en Tándem , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Eliminación de Gen , RNA-Seq , Biología Computacional , Secuencias de Aminoácidos , Regulación hacia Abajo , Técnicas In VitroRESUMEN
Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent.NEW & NOTEWORTHY Vasopressin is a key hormone that regulates the function of the collecting duct of the kidney. ERK1 and ERK2 are enzymes that play key roles in physiological regulation in all cells. The authors used collecting duct cell cultures to investigate the effects of vasopressin and the vasopressin receptor antagonist tolvaptan on ERK1 and ERK2 phosphorylation and activation.
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Sistema de Señalización de MAP Quinasas , Receptores de Vasopresinas , Tolvaptán/farmacología , Tolvaptán/metabolismo , Receptores de Vasopresinas/metabolismo , Fosforilación , Riñón/metabolismo , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Antagonistas de los Receptores de Hormonas Antidiuréticas/metabolismo , Vasopresinas/farmacología , Vasopresinas/metabolismoRESUMEN
We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.
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Desamino Arginina Vasopresina , Túbulos Renales Colectores , Ratones Noqueados , Animales , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Desamino Arginina Vasopresina/farmacología , Capacidad de Concentración Renal/efectos de los fármacos , Arginina Vasopresina/metabolismo , Masculino , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Ratones , Acuaporina 2/metabolismo , Acuaporina 2/genética , Fármacos Antidiuréticos/farmacología , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Ratones Endogámicos C57BL , Privación de Agua , Concentración Osmolar , Sodio/orina , Sodio/metabolismo , Vasopresinas/metabolismo , BenzazepinasRESUMEN
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.
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Acuaporina 2 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Ratones , Acuaporina 2/genética , beta Catenina/metabolismo , Biotina/metabolismo , Desamino Arginina Vasopresina/farmacología , Riñón/metabolismo , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño , Vasopresinas/farmacología , Vasopresinas/metabolismoRESUMEN
As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased â¼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by â¼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.
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Acuaporinas , Túbulos Renales Colectores , MicroARNs , Ratones , Masculino , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Cloruro de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acuaporinas/metabolismoRESUMEN
Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration methods to predict the responsible kinases for 51 phospho-proteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' Rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of seven independent data sets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A catalytic subunit-a (PKA), consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2d (CAMK2D). AMP-activated kinase catalytic subunit a-1 (PRKAA1) and CDC42 binding protein kinase beta (CDC42BPB).
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INTRODUCTION: Dimenhydrinate and scopolamine are frequently used drugs, but they cause drowsiness and performance decrement. Therefore, it is crucial to find peripheral targets and develop new drugs without central side effects. This study aimed to investigate the anti-motion sickness action and inner ear-related mechanisms of atrial natriuretic peptide (ANP). METHODS: Endolymph volume in the inner ear was measured with magnetic resonance imaging and expression of AQP2 and p-AQP2 was detected with Western blot analysis and immunofluorescence method. RESULTS: Both rotational stimulus and intraperitoneal arginine vasopressin (AVP) injection induced conditioned taste aversion (CTA) to 0.15% sodium saccharin solution and an increase in the endolymph volume of the inner ear. However, intraperitoneal injection of ANP effectively alleviated the CTA behaviour and reduced the increase in the endolymph volume after rotational stimulus. Intratympanic injection of ANP also inhibited rotational stimulus-induced CTA behaviour, but anantin peptide, an inhibitor of ANP receptor A (NPR-A), blocked this inhibitory effect of ANP. Both rotational stimulus and intraperitoneal AVP injection increased the expression of AQP2 and p-AQP2 in the inner ear of rats, but these increases were blunted by ANP injection. In in vitro experiments, ANP addition decreased AVP-induced increases in the expression and phosphorylation of AQP2 in cultured endolymphatic sac epithelial cells. CONCLUSION: Therefore, the present study suggests that ANP could alleviate motion sickness through regulating endolymph volume of the inner ear increased by AVP, and this action of ANP is potentially mediated by activating NPR-A and antagonising the increasing effect of AVP on AQP2 expression and phosphorylation.
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Arginina Vasopresina , Factor Natriurético Atrial , Endolinfa , Mareo por Movimiento , Animales , Factor Natriurético Atrial/farmacología , Factor Natriurético Atrial/metabolismo , Factor Natriurético Atrial/administración & dosificación , Arginina Vasopresina/farmacología , Arginina Vasopresina/administración & dosificación , Arginina Vasopresina/metabolismo , Mareo por Movimiento/tratamiento farmacológico , Masculino , Endolinfa/efectos de los fármacos , Endolinfa/metabolismo , Oído Interno/efectos de los fármacos , Ratas Sprague-Dawley , Acuaporina 2/metabolismo , RatasRESUMEN
AIMS: To discuss the role of autocrine/paracrine signaling of urothelial arginine vasopressin (AVP) on mammalian bladder capacities and micturition thresholds, impact of distension on water/urea reabsorption from the bladder, review of the literature to better characterize the central/peripheral effects of AVP, desmopressin (dAVP) toxicity, and urine biomarkers of nocturia. METHODS: This review summarizes discussions during an International Consultation on Incontinence-Research Society 2024 think tank with respect to the role of urothelial AVP in aged individuals with nocturnal polyuria, impact of solute and water reabsorption by the bladder on uninterrupted sleep, central effects of AVP, pharmacological basis of dAVP toxicity, and biomarkers in nocturia/lower urinary tract dysfunction (LUTD) with neurological diseases. RESULTS: Consensus recognized AVP function and pathways in the central nervous system (CNS), pre-proAVP localized using immunohistochemistry in bladder sections from adult/aged noncancerous human punch biopsies and rodent bladder sections is likely to accelerate the systemic uptake of water and urea from the bladder of anesthetized mice instilled with 3H-water and 14C-urea. Mechanisms for charged and uncharged solutes and water transport across the bladder, mechanism of dAVP toxicity, and utility of urine biomarkers in those with neurological diseases/nocturia were determined from literature reviews. CONCLUSION: Pre-proAVP is present in human/rodent bladders and may be involved in water reabsorption from bladder that prevents the sensation of fullness for uninterrupted sleep in healthy adults. The mechanism of action of AVP in the CNS was discussed, as was electrolyte/water transport across the bladder, the basis for dAVP toxicity, and feasibility of urine biomarkers to identify nocturia/LUTD with neurological diseases.
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The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.
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Acuaporina 2 , Acuaporina 5 , Proteínas del Citoesqueleto , Unión Proteica , Humanos , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Acuaporina 5/metabolismo , Acuaporina 5/química , Acuaporina 2/metabolismo , Acuaporina 2/química , Sitios de Unión , Acuaporinas/metabolismo , Acuaporinas/química , Dominios Proteicos , Modelos Moleculares , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/químicaRESUMEN
Background/aim: Nocturnal enuresis can be frustrating for children and their families as the child ages. Our aim is to evaluate urine aquaporin 2 (AQP-2) as a noninvasive biomarker of water balance in children with primary monosymptomatic nocturnal enuresis (PMNE). Material and methods: The study included 90 children; sixty-eight children suffering from PMNE aged (9.57 ± 2.16) years and 22 healthy children with good toilet control, matched sex and age. All enuretic children were subjected to complete history taking, clinical evaluation, and bed wetting diary. Serum arginine vasopressin (AVP) and urine AQP-2 were tested in the morning (at 9-11 am) and evening (at 9-11 pm). Blood urea, creatinine, Na, glucose, urine osmolality, Ca/Cr, Alb/Cr and specific gravity were tested simultaneously. Results: Serum AVP, urine AQP-2, and urine osmolality were statistically lower in patients than controls. Patients had a significantly lower level of night serum AVP concentrations, urine AQP-2, and urine osmolality than the corresponding morning level. Urine AQP-2 was significantly correlated with urine osmolality (p < 0.05). AQP-2 had a sensitivity of 90% and a specificity of 70%. However, no statistically significant correlation was found between serum AVP and urine AQP-2. Conclusion: Primary monosymptomatic nocturnal enuresis in children could be associated with reduction of urine excretion of AQP-2 at night. Urine AQP-2 is significantly correlated with urine osmolality. Therefore, it may be a noninvasive biomarker of hydration status in children with PMNE, with good sensitivity and specificity.
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Acuaporina 2 , Biomarcadores , Ritmo Circadiano , Enuresis Nocturna , Humanos , Niño , Enuresis Nocturna/orina , Enuresis Nocturna/sangre , Masculino , Femenino , Acuaporina 2/orina , Ritmo Circadiano/fisiología , Biomarcadores/orina , Biomarcadores/sangre , Concentración Osmolar , Estudios de Casos y Controles , Arginina Vasopresina/sangre , Arginina Vasopresina/orina , AdolescenteRESUMEN
Animal models of a variety of acquired nephrogenic diabetes insipidus (NDI) disorders have identified a common feature: all such models are associated with the loss of aquaporin-2 (AQP2) from collecting duct principal cells, explaining the associated polyuria. To discover mechanisms of AQP2 loss, previous investigators have carried out either transcriptomics (lithium-induced NDI, unilateral ureteral obstruction, endotoxin-induced NDI) or proteomics (hypokalaemia-associated NDI, hypercalcaemia-associated NDI, bilateral ureteral obstruction), yielding contrasting views. Here, to address whether there may be common mechanisms underlying loss of AQP2 in acquired NDI disorders, we have used bioinformatic data integration techniques to combine information from all transcriptomic and proteomic data sets. The analysis reveals roles for autophagy/apoptosis, oxidative stress and inflammatory signalling as key elements of the mechanism that results in loss of AQP2. These processes can cause AQP2 loss through the combined effects of repression of Aqp2 gene transcription, generalized translational repression, and increased autophagic degradation of proteins including AQP2. Two possible types of stress-sensor proteins, namely death receptors and stress-sensitive protein kinases of the EIF2AK family, are discussed as potential triggers for signalling processes that result in loss of AQP2. KEY POINTS: Prior studies have shown in a variety of animal models of acquired nephrogenic diabetes insipidus (NDI) that loss of the aquaporin-2 (AQP2) protein is a common feature. Investigations of acquired NDI using transcriptomics (RNA-seq) and proteomics (protein mass spectrometry) have led to differing conclusions regarding mechanisms of AQP2 loss. Bioinformatic integration of transcriptomic and proteomic data from these prior studies now reveals that acquired NDI models map to three core processes: oxidative stress, apoptosis/autophagy and inflammatory signalling. These processes cause loss of AQP2 through translational repression, accelerated degradation of proteins, and transcriptional repression.
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Body water homeostasis is maintained by the correct balance between water intake and water loss through urine, faeces, sweat and breath. It is known that elevated circulating levels of the antidiuretic hormone vasopressin decrease urine volume to prevent excessive water loss from the body. Vasopressin/cAMP/protein kinase A (PKA) signalling is the canonical pathway in renal collecting ducts for phosphorylating aquaporin-2 (AQP2) water channels, which leads to the reabsorption of water from urine via AQP2. Although recent omics data have verified various downstream targets of PKA, crucial regulators that mediate PKA-induced AQP2 phosphorylation remain unknown, mainly because vasopressin is usually used to activate PKA as a positive control. Vasopressin is extremely potent and phosphorylates various PKA substrates non-specifically, making it difficult to narrow down the candidate mediators responsible for AQP2 phosphorylation. The intracellular localization of PKA is tightly regulated by its scaffold proteins, also known as A-kinase anchoring proteins (AKAPs). Furthermore, each AKAP has a target domain that determines its intracellular localization, enabling the creation of a local PKA signalling network. Although vasopressin activates most PKAs independently of their intracellular localization, some chemical compounds preferentially act on PKAs localized on AQP2-containing vesicles while simultaneously phosphorylating AQP2 and its surrounding PKA substrates. Immunoprecipitation with antibodies against phosphorylated PKA substrates followed by mass spectrometry analysis revealed that the PKA substrate in proximity to AQP2 was lipopolysaccharide-responsive and beige-like anchor (LRBA). Furthermore, Lrba knockout studies revealed that LRBA was required for vasopressin-induced AQP2 phosphorylation.
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Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.
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Acuaporina 2 , Túbulos Renales Colectores , Ratas , Ratones , Animales , Acuaporina 2/metabolismo , Túbulos Renales Colectores/metabolismo , Fosforilación , Vasopresinas/farmacología , Vasopresinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas/metabolismo , Agua/metabolismoRESUMEN
Autophagy, a cellular process of "self-eating," plays an essential role in renal pathophysiology. However, the effect of autophagy on urine-concentrating ability in physiological conditions is still unknown. This study aimed to determine the relevance and mechanisms of autophagy for maintaining urine-concentrating capability during antidiuresis. The extent of the autophagic response to water deprivation (WD) was different between the renal cortex and medulla in mice. Autophagy activity levels in the renal cortex were initially suppressed and then stimulated by WD in a time-dependent manner. During 48 h WD, the urine-concentrating capability of mice was impaired by rapamycin (Rapa) but not by 3-methyladenine (3-MA), accompanied by suppressed renal aquaporin 2 (AQP2), V2 receptor (V2R), renin, and angiotensin-converting enzyme (ACE) expression, and levels of prorenin/renin, angiotensin II (ANG II), and aldosterone in the plasma and urine. In contrast, 3-MA and chloroquine (CQ) suppressed the urine-concentrating capability in WD72 mice, accompanied by downregulation of AQP2 and V2R expression in the renal cortex. 3-MA and CQ further increased AQP2 and V2R expression in the renal medulla of WD72 mice. Compared with 3-MA and CQ, Rapa administration yielded completely opposite results on the above parameters in WD72 mice. In addition, 3-MA and CQ abolished the upregulation of prorenin/renin, ANG II, and aldosterone levels in the plasma and urine in WD72 mice. Taken together, our study demonstrated that autophagy regulated urine-concentrating capability through differential regulation of renal AQP2/V2R and ACE/ANG II signaling during WD.NEW & NOTEWORTHY Autophagy exhibits a double-edged effect on cell survival and plays an essential role in renal pathophysiology. We for the first time reported a novel function of autophagy that controls the urine-concentrating capability in physiological conditions. We found that water deprivation (WD) differentially regulated autophagy in the kidneys of mice in a time-dependent manner and autophagy regulates the urine-concentrating capability mainly by regulating AQP2/V2R and ACE/ANG II signaling in the renal cortex in WD mice.
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Acuaporina 2 , Sistema Renina-Angiotensina , Animales , Ratones , Aldosterona , Angiotensina II , Autofagia , Cloroquina , Riñón , ReninaRESUMEN
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 (â¼2 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.
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Acuaporina 2 , Insulina , Animales , Masculino , Ratones , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Insulina/metabolismo , Mamíferos/metabolismo , Presión Osmótica , Vasopresinas/farmacología , Vasopresinas/metabolismo , Agua/metabolismoRESUMEN
Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-ß-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.
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Acuaporina 2 , Serina , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Células LLC-PK1 , Fosforilación , Serina/metabolismo , Porcinos , Vasopresinas/farmacología , Vasopresinas/metabolismo , Espacio Intracelular/metabolismoRESUMEN
The ability to regulate water movement is vital for the survival of cells and organisms. In addition to passively crossing lipid bilayers by diffusion, water transport is also driven across cell membranes by osmotic gradients through aquaporin water channels. There are 13 aquaporins in human tissues, and of these, aquaporin-2 (AQP2) is the most highly regulated water channel in the kidney: The expression and trafficking of AQP2 respond to body volume status and plasma osmolality via the antidiuretic hormone, vasopressin (VP). Dysfunctional VP signaling in renal epithelial cells contributes to disorders of water balance, and research initially focused on regulating the major cAMP/PKA pathway to normalize urine concentrating ability. With the discovery of novel and more complex signaling networks that regulate AQP2 trafficking, promising therapeutic targets have since been identified. Several strategies based on data from preclinical studies may ultimately translate to the care of patients with defective water homeostasis.
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Riñón/metabolismo , Desequilibrio Hidroelectrolítico/fisiopatología , Agua/metabolismo , Animales , Acuaporina 2/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Neurofisinas/metabolismo , Precursores de Proteínas/metabolismo , Transducción de Señal/fisiología , Vasopresinas/metabolismo , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
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 8 h 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.
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Diabetes Insípida Nefrogénica , Diabetes Mellitus , Túbulos Renales Colectores , Factores de Transcripción/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Arginina Vasopresina/metabolismo , Desamino Arginina Vasopresina/metabolismo , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/metabolismo , Diabetes Mellitus/metabolismo , Factor V/metabolismo , Túbulos Renales Colectores/metabolismo , Ratones , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/genética , Vasopresinas/metabolismo , Agua/metabolismoRESUMEN
BACKGROUND AND HYPOTHESIS: Tolvaptan, a vasopressin V2 receptor antagonist, is used for treating autosomal dominant polycystic kidney disease (ADPKD). We focused on changes in urinary osmolality (U-Osm) after tolvaptan initiation to determine whether they were associated with the therapeutic response to tolvaptan. METHODS: This was a single-centre, prospective, observational cohort study. Seventy-two patients with ADPKD who received tolvaptan were recruited. We analysed the relationship between changes in U-Osm and annual estimated glomerular filtration rate (eGFR) in terms of renal prognostic value using univariable and multivariable linear regression analyses. RESULTS: The mean value of U-Osm immediately before tolvaptan initiation was 351.8 ± 142.2 mosm/kg H2O, which decreased to 97.6 ± 23.8 mosm/kg H2O in the evening. The decrease in U-Osm was maintained in the outpatient clinic 1 month later. However, the values of U-Osm showed higher variability (160.2 ± 83.8 mosm/kg H2O) than did those in the first evening of tolvaptan administration. Multivariate analysis revealed that the baseline eGFR, baseline urinary protein, and U-Osm change in the evening of the day of admission (initial U-Osm drop) were significantly correlated with the subsequent annual change in eGFR. CONCLUSIONS: U-Osm can be measured easily and rapidly, and U-Osm change within a short time after tolvaptan initiation may be a useful index for the renal prognosis in actual clinical practice.
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BACKGROUND: The clinical utility of tolvaptan in chronic kidney disease (CKD) patients with heart failure remains uncertain. The level of urine cyclic adenosine monophosphate (AMP) relative to plasma arginine vasopressin (AVP) indicates the residual function of the collecting ducts in response to AVP stimulation and might be a key to predicting response of tolvaptan. METHODS: CKD patients who were hospitalized to treat their congestive heart failure refractory to conventional loop diuretics were considered to receive tolvaptan and included in this prospective study. The impact of urine cyclic AMP/plasma AVP ratio for prediction of response to tolvaptan, which was defined as any increase in urine volume at day 7 from day 0, was investigated. RESULTS: A total of 30 patients (median 75 years old, 24 men, and median estimated glomerular filtration rate 14.4 mL/min/1.73 m2) were included. As compared to baseline, urine volume increased at day 7 in 17 responders, whereas urine volume decreased at day 7 in 13 non-responders. Baseline urine cyclic AMP/plasma AVP ratio distributed between 0.25 and 4.01 with median 1.90. The urine cyclic AMP/plasma AVP ratio was a significant predictor of response to tolvaptan, which was adjusted for 6 potential confounders with a cutoff of 1.24. CONCLUSIONS: Baseline urine cyclic AMP/plasma AVP ratio is an independent predictor of response to tolvaptan in advanced CKD patients with heart failure. CLINICAL TRIAL REGISTRATION: UMIN000022422.