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1.
J Cell Mol Med ; 28(8): e18301, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38652212

RESUMO

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the ß3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the ß3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the ß3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human ß3-AR agonists might represent an effective possible treatment strategy for X-NDI.


Assuntos
Agonistas de Receptores Adrenérgicos beta 3 , Masculino , Animais , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Agonistas de Receptores Adrenérgicos beta 3/uso terapêutico , Antidiuréticos/farmacologia , Antidiuréticos/uso terapêutico , Capacidade de Concentração Renal/efeitos dos fármacos , Polidipsia/tratamento farmacológico , Polidipsia/etiologia
2.
Glia ; 72(5): 938-959, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38362923

RESUMO

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.


Assuntos
Astrócitos , Canais de Cátion TRPV , Astrócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Aquaporina 4/metabolismo , Neuroglia/metabolismo , Encéfalo/metabolismo
3.
EMBO J ; 39(17): e105696, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32716134

RESUMO

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Núcleo Celular/genética , Retículo Endoplasmático/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/genética , Camundongos , Oryzias
4.
J Transl Med ; 21(1): 340, 2023 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-37217929

RESUMO

BACKGROUND: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. METHODS: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. RESULTS: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. CONCLUSIONS: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.


Assuntos
Lamina Tipo A , Miócitos Cardíacos , Humanos , Apoptose , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Guanabenzo/farmacologia , Homeostase , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Miócitos Cardíacos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Resposta a Proteínas não Dobradas
5.
Nature ; 542(7641): 362-366, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28178232

RESUMO

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.


Assuntos
Mesoderma/patologia , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Estresse do Retículo Endoplasmático/genética , Feminino , Genes myc , Genes ras , Humanos , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Mesoderma/metabolismo , Camundongos , Mosaicismo , Proteína Oncogênica p55(v-myc)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/metabolismo , Transcriptoma/genética , Gencitabina
6.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674662

RESUMO

We previously reported the novel finding that ß3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of ß3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the ß3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.


Assuntos
Diabetes Insípido Nefrogênico , Diabetes Mellitus , Camundongos , Animais , Humanos , Diabetes Insípido Nefrogênico/tratamento farmacológico , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/metabolismo , Aquaporina 2/genética , Aquaporina 2/metabolismo , Poliúria/tratamento farmacológico , Agonistas Adrenérgicos beta
7.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675161

RESUMO

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.


Assuntos
Aquaporina 2 , Túbulos Renais Coletores , Lisossomos , Água , Animais , Camundongos , Aquaporina 2/genética , Aquaporina 2/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Macrolídeos/farmacologia , Macrolídeos/metabolismo , Água/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo
8.
J Cell Mol Med ; 25(23): 10902-10915, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34773379

RESUMO

Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies strictly associated with significant deterioration of the electrical and contractile function of the heart. Despite the continuous flow of biomedical evidence, linking cardiac inflammation to heart remodelling in patients harbouring lmna mutations is puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belonging to four different families carrying pathogenic lmna mutations segregating with cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n = 11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6) were found in all affected patients' sera. In addition, elevated levels of Interleukins (IL) IL-1Ra, IL-1ß IL-4, IL-5 and IL-8 and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured in a large subset of patients associated with more aggressive clinical manifestations. Finally, the expression of the pro-inflammatory 70 kDa heat shock protein (Hsp70) was significantly increased in serum exosomes of patients harbouring the lmna mutation associated with the more severe phenotype. Overall, the identification of patient subsets with overactive or dysregulated myocardial inflammatory responses could represent an innovative diagnostic, prognostic and therapeutic tool against Lamin A/C cardiomyopathies.


Assuntos
Citocinas/metabolismo , Cardiopatias/metabolismo , Inflamação/metabolismo , Adulto , Cardiolipinas/metabolismo , Linhagem Celular , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/metabolismo
9.
Glia ; 69(9): 2235-2251, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34038017

RESUMO

In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin-4 (M1-AQP4 and M23-AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23-null mouse model (M23-null). In the M23-null spinal cord OAP assembly, the perivascular localization of AQP4 and M1-AQP4 protein were drastically reduced. In heterozygous, M1-AQP4 was proportionally reduced with M23-AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23-AQP4 in the regulation of M1-AQP4 expression. M1-AQP4 transcription, splicing and M1-AQP4 protein degradation were found to be unaffected in M23-null spinal cord and in M23-null astrocyte primary culture. The translational control was investigated by mRNA-protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4-RBPs). Compared to WT, in M23-null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4-RBPs and provide evidence that in mouse spinal cord M23-AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.


Assuntos
Aquaporina 4 , Astrócitos , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Membrana Celular/metabolismo , Sistema Nervoso Central/metabolismo , Camundongos , Isoformas de Proteínas
10.
Glia ; 69(2): 473-488, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32946135

RESUMO

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel "OAP-null" mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.


Assuntos
Astrócitos , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Camundongos , Camundongos Knockout , Isoformas de Proteínas/metabolismo
11.
Nature ; 528(7581): 272-5, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26595272

RESUMO

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.


Assuntos
Autofagia/fisiologia , Desenvolvimento Ósseo/fisiologia , Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais , Animais , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Desenvolvimento Ósseo/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Embrião de Mamíferos , Matriz Extracelular/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo
12.
Int J Mol Sci ; 22(22)2021 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-34830416

RESUMO

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.


Assuntos
Aquaporina 1/genética , Transporte Biológico/genética , Epitélio/metabolismo , Peritônio/metabolismo , Aquaporinas/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Diálise Peritoneal/normas , Peritônio/patologia , Sódio/metabolismo
13.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374405

RESUMO

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end-stage renal disease (ESRD) lies in the fact that the currently used glucose-based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and l-carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro-inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro-inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility.


Assuntos
Carnitina/química , Soluções para Diálise/química , Epitélio/metabolismo , Glucose/metabolismo , Diálise Peritoneal/métodos , Bicarbonatos/farmacologia , Materiais Biocompatíveis , Sobrevivência Celular , Citocinas/metabolismo , Humanos , Inflamação , Falência Renal Crônica , Microscopia Confocal , Peritônio/efeitos dos fármacos , Junções Íntimas/metabolismo , Ultrafiltração , Xilitol/química
14.
J Cell Mol Med ; 23(9): 6331-6342, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31361068

RESUMO

The K+ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K+ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy.


Assuntos
Canal de Potássio ERG1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Mutação/genética , Adolescente , Adulto , Linhagem Celular , Membrana Celular/genética , Criança , Eletrocardiografia/métodos , Feminino , Células HEK293 , Humanos , Masculino , Fenótipo , Transporte Proteico/genética , Adulto Jovem
15.
J Cell Mol Med ; 23(2): 1034-1049, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30426662

RESUMO

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvß3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvß3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvß3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Estreptozocina/farmacologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Inflamação/metabolismo , Rim/metabolismo , Masculino , Podócitos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
16.
J Cell Mol Med ; 22(2): 1236-1246, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29055082

RESUMO

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO-IgG) against the water channel aquaporin-4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle. This conclusion was obtained using human, rat and mouse skeletal muscle including the AQP4-KO mouse. A biochemical analysis using a new size exclusion chromatography approach for AQP4 suprastructure fractionation revealed substantial differences in supramolecular AQP4 assemblies and isoform abundance between brain and skeletal muscle matching a lower binding affinity of NMO-IgG to muscle compared to the brain. Super-resolution microscopy analysis with g-STED revealed different AQP4 organization in native tissues, while in the brain perivascular astrocyte endfoot membrane AQP4 was mainly organized in large interconnected and raft-like clusters, in the sarcolemma of fast-twitch fibres AQP4 aggregates often appeared as small, relatively isolated linear entities. In conclusion, our results provide evidence that AQP4 supramolecular structure is different in brain and skeletal muscle, which is likely to result in different tissues susceptibility to the NMO disease.


Assuntos
Aquaporina 4/química , Aquaporina 4/metabolismo , Encéfalo/metabolismo , Músculos/metabolismo , Neuromielite Óptica/metabolismo , Agregados Proteicos , Animais , Cromatografia em Gel , Humanos , Imunoglobulina G/metabolismo , Ligação Proteica , Ratos Wistar
17.
J Cell Mol Med ; 22(2): 904-912, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29044946

RESUMO

Aquaporin-1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis-dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF-1α, caspase-3 (CASP3) and metalloproteinase-2 (MMP2) protein levels. We found that AQP1 knock-down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA-treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1-dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF-1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.


Assuntos
Aquaporina 1/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Animais , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , Modelos Animais de Doenças , Inativação Gênica , Neoplasias Pulmonares/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Melanoma Experimental/irrigação sanguínea , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Fatores de Tempo , Hipóxia Tumoral
18.
J Cell Mol Med ; 22(1): 613-627, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28940930

RESUMO

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia.


Assuntos
Aquaporina 4/deficiência , Metilação de DNA/genética , Hipóxia/genética , Oxigênio/efeitos adversos , Doenças Retinianas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Aquaporina 4/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ilhas de CpG/genética , Eletrorretinografia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Knockout , Modelos Biológicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Retina/metabolismo , Retina/patologia , Doenças Retinianas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cell Physiol Biochem ; 48(2): 847-862, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30032151

RESUMO

BACKGROUND/AIMS: We recently showed that the ß3-adrenoreceptor (ß3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a ß3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic ß3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human ß3AR. METHODS: Human ß3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect ß3AR signaling under acute and chronic stimulation. Plasma membrane localization of ß3AR, AVPR2 and AQP2 after agonist stimulation was studied by confocal microscopy. Receptors degradation was evaluated by Western blotting. RESULTS: In renal cells acute stimulation with the selective ß3AR agonist mirabegron, induced a dose-dependent increase in cAMP. Interestingly, chronic exposure to mirabegron promoted a significant increase of intracellular cAMP up to 12 hours. In addition, a slow and slight agonist-induced internalization and a delayed downregulation of ß3AR was observed under chronic stimulation. Furthermore, chronic exposure to mirabegron promoted apical expression of AQP2 also up to 12 hours. Conversely, long-term stimulation of AVPR2 with dDAVP showed short-lasting receptor signaling, rapid internalization and downregulation and apical AQP2 expression for no longer than 3 h. CONCLUSIONS: Overall, we conclude that ß3AR is less prone than AVPR2 to agonist-induced desensitization in renal collecting duct epithelial cells, showing sustained cAMP production, preserved membrane localization and delayed degradation after 12 hours agonist exposure. These results may be important for the potential use of chronic pharmacological stimulation of ß3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2.


Assuntos
Acetanilidas/farmacologia , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Receptores Adrenérgicos beta 3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Aquaporina 2/metabolismo , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Túbulos Renais Coletores/citologia , Camundongos , Microscopia Confocal , Receptores Adrenérgicos beta 3/química , Receptores de Vasopressinas/metabolismo
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