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1.
Genes Chromosomes Cancer ; 62(7): 377-391, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36562080

RESUMO

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.


Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Proliferação de Células/genética , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genética
2.
Front Cell Dev Biol ; 10: 919438, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35874817

RESUMO

Vasopressin (AVP) plays a key function in controlling body water and salt balance through the activation of the vasopressin receptors V1aR and V2R. Abnormal secretion of AVP can cause the syndrome of inappropriate antidiuresis that leads to hyponatremia, which is an electrolyte disorder often observed in the elderly hospitalized and oncologic patients. Beyond kidneys, the colonic epithelium modulates water and salt homeostasis. The water channel AQP3, expressed in villus epithelial cells is implicated in water absorption across human colonic surface cells. Here, the action of dDAVP, a stable vasopressin analog, was evaluated on the AQP3 expression and function using human colon HCT8 cells as an experimental model. Confocal and Western Blotting analysis revealed that HCT8 cells express both V1aR and V2R. Long-term (72 h) treatment with dDAVP reduced glycerol uptake and cell viability. These effects were prevented by SR49059, a synthetic antagonist of V1aR, but not by tolvaptan, a specific V2R antagonist. Of note, the SR49059 action was impaired by DFP00173, a selective inhibitor of AQP3. Interestingly, compared to the normal colonic mucosa, in the colon of patients with adenocarcinoma, the expression of V1aR was significantly decreased. These findings were confirmed by gene expression analysis with RNA-Seq data. Overall, data suggest that dDAVP, through the V1aR dependent pathway, reduces AQP3 mediated glycerol uptake, a process that is reversed in adenocarcinoma, suggesting that the AVP-dependent AQP3 pathway may represent a novel target in colon diseases associated with abnormal cell growth.

3.
Sci Rep ; 11(1): 4537, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33633156

RESUMO

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.


Assuntos
Aquaporina 2/metabolismo , Olea/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Receptores de Detecção de Cálcio/agonistas , Vasopressinas/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Extratos Vegetais/química , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores de Detecção de Cálcio/metabolismo
4.
Foods ; 10(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374501

RESUMO

The production of olive oil is accompanied by the generation of a huge amount of waste and by-products including olive leaves, pomace, and wastewater. The latter represents a relevant environmental issue because they contain certain phytotoxic compounds that may need specific treatments before the expensive disposal. Therefore, reducing waste biomass and valorizing by-products would make olive oil production more sustainable. Here, we explore the biological actions of extracts deriving from olive by-products including olive pomace (OP), olive wastewater (OWW), and olive leaf (OLs) in human colorectal carcinoma HCT8 cells. Interestingly, with the same phenolic concentration, the extract obtained from the OWW showed higher antioxidant ability compared with the extracts derived from OP and OLs. These biological effects may be related to the differential phenolic composition of the extracts, as OWW extract contains the highest amount of hydroxytyrosol and tyrosol that are potent antioxidant compounds. Furthermore, OP extract that contains a higher level of vanillic acid than the other extracts displayed a cytotoxic action at the highest concentration. Together these findings revealed that phenols in the by-product extracts may interfere with signaling molecules that cross-link several intracellular pathways, raising the possibility to use them for beneficial health effects.

5.
Cells ; 9(6)2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32486031

RESUMO

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13-Rho-ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.


Assuntos
Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Síndrome de Secreção Inadequada de HAD/genética , Mutação/genética , Fosfosserina/metabolismo , Receptores de Vasopressinas/genética , Transdução de Sinais , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Osmose , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Água/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
6.
Antioxidants (Basel) ; 9(3)2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32214012

RESUMO

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties.

7.
PLoS One ; 14(3): e0214159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897184

RESUMO

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 µM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity.


Assuntos
Cádmio/toxicidade , Rim/efeitos dos fármacos , Olea , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Rim/citologia , Camundongos , Olea/química , Extratos Vegetais/química , Substâncias Protetoras/química
8.
Front Mol Biosci ; 5: 77, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30197885

RESUMO

Autosomal Dominant Polycistic kidney Disease (ADPKD) is a renal channelopathy due to loss-of-function mutations in the PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a large protein found predominantly on the plasma membrane where interacts with different proteins, including PC2. PC2 is a smaller integral membrane protein also expressed in intracellular organelles, acting as a non-selective cation channel permeable to calcium. Both PC1 and PC2 are also localized to the primary cilium of renal epithelial cells serving as mechanosensor that controls calcium influx through the plasma membrane and regulates intracellular calcium release from the endoplasmic reticulum. The mechanisms by which PC1/2 dysfunction leads to ADPKD needs still to be clarified. We have recently reported that selective Calcium-Sensing Receptor (CaSR) activation in human conditionally immortalized Proximal Tubular Epithelial cells deficient for PC1 (ciPTEC-PC1KD), deriving from urine sediments reduces intracellular cAMP and mTOR activity, and increases intracellular calcium reversing the principal ADPKD dysregulations. Reduced cellular free calcium found in ADPKD can, on the other hand, affect mitochondrial function and ATP production and, interestingly, a relationship between mitochondria and renal polycystic diseases have been suggested. By using ciPTEC-PC1KD as experimental tool modeling of ADPKD, we show here that, compared with wild type cells, ciPTEC-PC1KD have significantly lower mitochondrial calcium levels associated with a severe deficit in mitochondrial ATP production, secondary to a multilevel impairment of oxidative phosphorylation. Notably, selective CaSR activation with the calcimimetic NPS-R568 increases mitochondrial calcium content close to the levels found in resting wild type cells, and fully recovers the cell energy deficit associated to the PC1 channel disruption. Treatment of ciPTEC-PC1KD with 2-APB, an IP3R inhibitor, prevented the rescue of bioenergetics deficit induced by CaSR activation supporting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle. Together these data indicate that, besides reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, selective CaSR activation in PKD1 deficient cells restores altered mitochondrial function that, in ADPKD, is known to facilitate cyst formation. These findings identify CaSR as a potential therapeutic target.

9.
Sci Rep ; 8(1): 5704, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29632324

RESUMO

Clinical and fundamental research suggest that altered calcium and cAMP signaling might be the most proximal events in ADPKD pathogenesis. Cells from ADPKD cysts have a reduced resting cytosolic calcium [Ca2+]i and increased cAMP levels. CaSR plays an essential role in regulating calcium homeostasis. Its activation is associated with [Ca2+]i increase and cAMP decrease, making CaSR a possible therapeutic target. Human conditionally immortalized Proximal Tubular Epithelial cells (ciPTEC) with stable knockdown of PKD1 (ciPTEC-PC1KD) and ciPTEC generated from an ADPKD1 patient (ciPTEC-PC1Pt) were used as experimental tools. CaSR functional expression was confirmed by studies showing that the calcimimetic NPS-R568 induced a significant increase in [Ca2+]i in ciPTEC-PC1KD and ciPTEC-PC1Pt. Resting [Ca2+]i were significantly lower in ciPTEC-PC1KD with respect to ciPTECwt, confirming calcium dysregulation. As in native cyst cells, significantly higher cAMP levels and mTOR activity were found in ciPTEC-PC1KD compared to ciPTECwt. Of note, NPS-R568 treatment significantly reduced intracellular cAMP and mTOR activity in ciPTEC-PC1KD and ciPTEC-PC1Pt. To conclude, we demonstrated that selective CaSR activation in human ciPTEC carrying PKD1 mutation increases [Ca2+]i, reduces intracellular cAMP and mTOR activity, reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, making CaSR a possible candidate as therapeutic target.


Assuntos
Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Canais de Cátion TRPP/genética , Células Cultivadas , AMP Cíclico/metabolismo , Citosol/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Túbulos Renais Proximais/citologia , Mutação , Fenetilaminas/farmacologia , Propilaminas/farmacologia , Serina-Treonina Quinases TOR/metabolismo
10.
Cell Mol Life Sci ; 75(18): 3411-3422, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29549422

RESUMO

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.


Assuntos
AMP Cíclico/metabolismo , Cistinose/patologia , Mitocôndrias/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Linhagem Celular , Cistinose/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Potencial da Membrana Mitocondrial , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sirtuína 3/metabolismo
11.
Cell Physiol Biochem ; 44(2): 515-531, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29145196

RESUMO

BACKGROUND/AIMS: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. METHODS: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. RESULTS: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. CONCLUSION: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.


Assuntos
Aquaporina 2/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Aquaporina 2/genética , Benzoquinonas/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Endocitose , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Imunoprecipitação , Rim/metabolismo , Rim/patologia , Lactamas Macrocíclicas/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética
12.
Pflugers Arch ; 469(9): 1163-1176, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28378089

RESUMO

Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.


Assuntos
Actinas/metabolismo , Brônquios/metabolismo , Interleucina-13/metabolismo , Transdução de Sinais/fisiologia , Transportadores de Sulfato/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Asma/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Quinases Associadas a rho/metabolismo
13.
J Cell Mol Med ; 21(9): 1767-1780, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28326667

RESUMO

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin-regulated water reabsorption via aquaporin-2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2-mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin-cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP-induced increase in ser256-AQP2 and osmotic water permeability. A similar effect on ser256-AQP2 was found in V1aR -/- mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan-V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256-AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium-inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.


Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Aquaporina 2/metabolismo , Benzazepinas/farmacologia , Cálcio/metabolismo , Citosol/metabolismo , Receptores de Vasopressinas/metabolismo , Idoso de 80 Anos ou mais , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Aquaporina 2/urina , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Cães , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Síndrome de Secreção Inadequada de HAD/sangue , Síndrome de Secreção Inadequada de HAD/tratamento farmacológico , Rim/efeitos dos fármacos , Rim/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Osmose , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Transporte Proteico/efeitos dos fármacos , Sódio/sangue , Tolvaptan , Água/metabolismo
14.
J Cell Sci ; 128(13): 2350-60, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25977473

RESUMO

We previously described that high luminal Ca(2+) in the renal collecting duct attenuates short-term vasopressin-induced aquaporin-2 (AQP2) trafficking through activation of the Ca(2+)-sensing receptor (CaSR). Here, we evaluated AQP2 phosphorylation and permeability, in both renal HEK-293 cells and in the dissected inner medullary collecting duct, in response to specific activation of CaSR with NPS-R568. In CaSR-transfected cells, CaSR activation drastically reduced the basal levels of AQP2 phosphorylation at S256 (AQP2-pS256), thus having an opposite effect to vasopressin action. When forskolin stimulation was performed in the presence of NPS-R568, the increase in AQP2-pS256 and in the osmotic water permeability were prevented. In the freshly isolated inner mouse medullar collecting duct, stimulation with forskolin in the presence of NPS-R568 prevented the increase in AQP2-pS256 and osmotic water permeability. Our data demonstrate that the activation of CaSR in the collecting duct prevents the cAMP-dependent increase in AQP2-pS256 and water permeability, counteracting the short-term vasopressin response. By extension, our results suggest the attractive concept that CaSR expressed in distinct nephron segments exerts a negative feedback on hormones acting through cAMP, conferring high sensitivity of hormone to extracellular Ca(2+).


Assuntos
Aquaporina 2/metabolismo , Cálcio/farmacologia , Espaço Extracelular/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasopressinas/farmacologia , Adenilil Ciclases/metabolismo , Compostos de Anilina/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos , Osmose/efeitos dos fármacos , Fenetilaminas , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Propilaminas , Ratos , Espalhamento de Radiação , Água/metabolismo
15.
Am J Physiol Renal Physiol ; 308(11): F1200-6, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25656364

RESUMO

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, which plays an essential role in regulating Ca(2+) homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Immunolocalization studies of polarized ciPTEC revealed the apical localization of the receptor. By Western blotting of ciPTEC lysates, both monomeric and dimeric forms of CaSR at 130 and ∼250 kDa, respectively, were detected. Functional studies indicated that both external calcium and the positive CaSR allosteric modulator, NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to its agonists. Calcium depletion from the endoplasmic reticulum using cyclopiazonic acid abolished the increase in cytosolic calcium elicited by NPS-R568, confirming calcium exit from intracellular stores. Activation of CaSR by NPS-R significantly reduced the increase in cAMP elicited by forskolin (FK), a direct activator of adenylate cyclase, further confirming the functional expression of the receptor in this cell line. CaSR expressed in ciPTEC was found to interact with Gq as a downstream effector, which in turn can cause release of calcium from intracellular stores via phospholipase C activation. We conclude that human proximal tubular ciPTEC express functional CaSR and respond to its activation with a release of calcium from intracellular stores. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Compostos de Anilina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Separação Celular/métodos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Fenetilaminas , Propilaminas , Receptores de Detecção de Cálcio/análise
16.
J Biol Chem ; 289(40): 27807-13, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25112872

RESUMO

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.


Assuntos
Aquaporina 2/metabolismo , Glutationa/metabolismo , Processamento de Proteína Pós-Traducional , Água/metabolismo , Animais , Aquaporina 2/genética , Células HEK293 , Humanos , Rim/metabolismo , Camundongos , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
17.
J Am Soc Nephrol ; 25(10): 2241-53, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24700872

RESUMO

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.


Assuntos
Aquaporina 2/metabolismo , Sinalização do Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Doenças Renais Policísticas/enzimologia , Proteína Fosfatase 2/metabolismo , Animais , Quinases Ciclina-Dependentes/antagonistas & inibidores , Haploinsuficiência , Técnicas In Vitro , Masculino , Camundongos , Doenças Renais Policísticas/genética , Purinas , Ratos Sprague-Dawley , Roscovitina , Vasopressinas/metabolismo
18.
PLoS One ; 8(11): e79113, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244430

RESUMO

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.


Assuntos
Sinalização do Cálcio , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Mutação de Sentido Incorreto , Receptores de Detecção de Cálcio/metabolismo , Substituição de Aminoácidos , Retículo Endoplasmático/genética , Células HEK293 , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de Detecção de Cálcio/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
19.
Mol Pharm ; 10(12): 4620-8, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24168213

RESUMO

Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Purinas/farmacologia , Animais , Linhagem Celular , Citosol/efeitos dos fármacos , Citosol/metabolismo , Cães , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Indóis/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Células Madin Darby de Rim Canino , Roscovitina , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
20.
Cell Physiol Biochem ; 32(7): 184-99, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24429825

RESUMO

BACKGROUND: We recently reported that aquaporin 5 (AQP5), a water channel never identified in the kidney before, co-localizes with pendrin at the apical membrane of type-B intercalated cells in the kidney cortex. Since co-expression of AQP5 and pendrin in the apical membrane domain is a common feature of several other epithelia such as cochlear and bronchial epithelial cells, we evaluated here whether this strict membrane association may reflect a co-regulation of the two proteins. To investigate this possibility, we analyzed AQP5 and pendrin expression and trafficking in mice under chronic K(+) depletion, a condition that results in an increased ability of renal tubule to reabsorb bicarbonate, often leads to metabolic alkalosis and is known to strongly reduce pendrin expression. METHODS: Mice were housed in metabolic cages and pair-fed with either a standard laboratory chow or a K(+)-deficient diet. AQP5 abundance was assessed by western blot in whole kidney homogenates and AQP5 and pendrin were localized by confocal microscopy in kidney sections from those mice. In addition, the short-term effect of changes in external pH on pendrin trafficking was evaluated by fluorescence resonance energy transfer (FRET) in MDCK cells, and the functional activity of pendrin was tested in the presence and absence of AQP5 in HEK 293 Phoenix cells. RESULTS: Chronic K(+) depletion caused a strong reduction in pendrin and AQP5 expression. Moreover, both proteins shifted from the apical cell membrane to an intracellular compartment. An acute pH shift from 7.4 to 7.0 caused pendrin internalization from the plasma membrane. Conversely, a pH shift from 7.4 to 7.8 caused a significant increase in the cell surface expression of pendrin. Finally, pendrin ion transport activity was not affected by co-expression with AQP5. CONCLUSIONS: The co-regulation of pendrin and AQP5 membrane expression under chronic K(+)-deficiency indicates that these two molecules could cooperate as an osmosensor to rapidly detect and respond to alterations in luminal fluid osmolality.


Assuntos
Proteínas de Transporte de Ânions/biossíntese , Aquaporina 5/biossíntese , Córtex Renal/metabolismo , Potássio/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Aquaporina 5/metabolismo , Bicarbonatos/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/genética , Córtex Renal/citologia , Camundongos , Microscopia Imunoeletrônica , Transportadores de Sulfato
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