RESUMEN
In endothelial cells, erythropoietin receptors (EPORs) mediate the protective, proliferative and angiogenic effects of EPO and its analogues, which act as EPOR agonists. Because hormonal receptors undergo functional changes upon chronic exposure to agonists and because erythropoiesis-stimulating agents (ESAs) are used for the long-term treatment of anemia, it is critical to determine the mechanism by which EPOR responsiveness is regulated at the vascular level after prolonged exposure to ESAs. Here, we investigated EPOR desensitization/resensitization in human umbilical vein endothelial cells (HUVECs) upon exposure to three ESAs with different pharmacokinetic profiles, epoetin alpha (EPOα), darbepoetin alpha (DarbEPO) and continuous EPOR activator (CERA). These agonists all induced activation of the transcription factor STAT-5, which is a component of the intracellular pathway associated with EPORs. STAT-5 activation occurred with either monophasic or biphasic kinetics for EPOα/DarbEPO and CERA, respectively. ESAs, likely through activation of the STAT-5 pathway, induced endothelial cell proliferation and stimulated angiogenesis in vitro, demonstrating a functional role for epoetins on endothelial cells. All epoetins induced EPOR desensitization with more rapid kinetics for CERA compared to EPOα and DarbEPO. However, the recovery of receptor responsiveness was strictly dependent on the type of epoetin, the agonist concentration and the time of exposure to the agonist. EPOR resensitization occurred with more rapid kinetics after exposure to low epoetin concentrations for a short period of desensitization. When the highest concentration of agonists was tested, the recovery of receptor responsiveness was more rapid with CERA compared to EPOα and was completely absent with DarbEPO. Our results demonstrate that these three ESAs regulate EPOR resensitization by very different mechanisms and that both the type of molecule and the length of EPOR stimulation are factors that are critical for the control of EPOR functioning in endothelial cells. The differences observed in receptor resensitization after stimulation with the structurally different ESAs are most likely due different control mechanisms of receptor turnover at the intracellular level.
RESUMEN
Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D(2)-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D(2)-like potency comparable to that of dopamine.
Asunto(s)
Benzofuranos/farmacología , Imidazoles/farmacología , Receptores de Dopamina D2/agonistas , Animales , Benzofuranos/síntesis química , Benzofuranos/química , Sitios de Unión , Imidazoles/síntesis química , Imidazoles/química , Ligandos , Masculino , Modelos Moleculares , Estructura Molecular , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Obesity is a chronic low inflammatory state. In the obesity condition the white adipose tissue (WAT) is massively infiltrated with monocytes/macrophages, and the nature of the signals recruiting these inflammatory cells has yet to be fully elucidated. Haptoglobin (Hp) is an inflammatory marker and its expression is induced in the WAT of obese subjects. In an effort to elucidate the biological significance of Hp presence in the WAT and of its upregulation in obesity we formulated the hypothesis that Hp may serve as a macrophage chemoattractant. RESULTS: We demonstrated by chemotaxis assay that Hp is able to attract chemokine (C-C motif) receptor 2 (CCR2)-transfected pre-B lymphocytes and monocytes in a dose-dependent manner. Moreover, Hp-mediated migration of monocytes is impaired by CCR2-specific inhibition or previous cell exposure to monocyte chemoattractant protein 1 (MCP1) (also known as CCR2 ligand or chemokine (C-C motif) ligand 2 (CCL2)). Downstream effects of Hp/CCR2 interaction were also investigated: flow cytometry proved that monocytes treated with Hp show reduced CCR2 expression on their surface; Hp interaction induces calcium release that is reduced upon pretreatment with CCR2 antagonist; extracellular signal-regulated kinase (ERK)1/2, a signal transducer activated by CCR2, is phosphorylated following Hp treatment and this phosphorylation is reduced when cells are pretreated with a specific CCR2 inhibitor. Consistently, blocking the ERK1/2 pathway with U0126, the selective inhibitor of the ERK upstream mitogen-activated protein (MAP)-ERK kinase (MEK), results in a dramatic reduction (by almost 100%) of the capability of Hp to induce monocyte migration. CONCLUSIONS: Our data show that Hp is a novel monocyte chemoattractant and that its chemotactic potential is mediated, at least in part. by its interaction with CCR2.
Asunto(s)
Quimiotaxis/fisiología , Haptoglobinas/metabolismo , Monocitos/fisiología , Receptores CCR2/metabolismo , Adulto , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Butadienos/farmacología , Calcio/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimiocina CCL2/metabolismo , Quimiotaxis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Receptores CCR2/agonistas , Receptores CCR2/antagonistas & inhibidores , Células U937 , Adulto JovenRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can lead to kidney failure resulting in end-stage renal disease. ADPKD is mainly caused by mutations in either the PKD1 and PKD2 genes, encoding for polycystin-1 and polycystin-2, respectively. In order to clarify the disease mechanisms, here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which the PKD2 gene was deleted using CRISPR/Cas9 technology. The PKD2-/- iPSCs expressed the main pluripotency markers, were able to differentiate into the three germ layers and had a normal karyotype.
Asunto(s)
Sistemas CRISPR-Cas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Canales Catiónicos TRPP/genética , Línea Celular , Humanos , MutaciónRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, characterised by the development of multiple fluid-filled cysts in the kidneys and other organs. PKD1 and PKD2 are the two major causative genes encoding for polycystin-1 and polycystin-2, respectively. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines with either heterozygous or compound heterozygous mutations in the PKD1 gene using CRISPR-Cas9 technology. The PKD1+/- and PKD1-/- iPSCs maintain stem cell-like morphology, normal karyotype, pluripotency and differentiation capacity in the three germ layers.
RESUMEN
2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A(1) adenosine receptor, and many of them also show a good selectivity for A(1) with respect to A(2A) and A(3) adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A(1) adenosine receptor.
Asunto(s)
Antagonistas del Receptor de Adenosina A1 , Purinas/síntesis química , Purinas/farmacología , Antagonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A3 , Animales , Cinética , Ligandos , Purinas/química , Relación Estructura-Actividad Cuantitativa , Ratas , Receptor de Adenosina A1/química , Receptor de Adenosina A2A/química , Receptor de Adenosina A3/químicaRESUMEN
Individuals with a diagnosis of adult separation anxiety (ASAD) have extreme anxiety about separations, actual or imagined, from major attachment figures. ASAD might represent a psychological/behavioral model for research probably involving a dysregulation of those neurobiological mechanisms of attachment, in particular central oxytocin (OT), described in numerous animal studies. As experimental strategy, we chose the nucleotidic sequencing of the human OT gene of patients with ASAD to evaluate whether OT mutations were related to potential alteration of its production. With this aim, mutation scanning of proximal promoter and untranslated and coding regions of the OT gene was carried out in 36 patients with ASAD, 14 patients without ASAD, and 26 controls. No mutations were found in promoter and coding regions of the OT gene in our population. One rare 3'UTR single nucleotide variant (rs17339677) and one intron 2 molecular variant (rs34097556), which showed a high frequency, were evidenced. There was no significant difference in the genotype distribution of this intron 2 polymorphism between patients and healthy individuals. Further research is needed to investigate the association between ASAD and OT peptide and receptor polymorphisms.
Asunto(s)
Ansiedad de Separación/genética , Mutación/genética , Oxitócicos , Oxitocina/genética , Regiones no Traducidas 3' , Adulto , Factores de Edad , Edad de Inicio , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Variación Genética , Genotipo , Humanos , Intrones , Masculino , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/terapia , Factor de Transcripción PAX2/genética , Adulto , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ingeniería Genética/métodos , Mutación de Línea Germinal/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Glomérulos Renales/metabolismo , Mutación/genética , Factor de Transcripción PAX2/análisis , Podocitos/química , Podocitos/metabolismo , Podocitos/fisiología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Generating new kidneys using tissue engineering technologies is an innovative strategy for overcoming the shortage of donor organs for transplantation. Here we report how to efficiently engineer the kidney vasculature of decellularized rat kidney scaffolds by using human induced pluripotent stem cell (hiPSCs)-derived endothelial cells (hiPSC-ECs). In vitro, hiPSC-ECs responded to flow stress by acquiring an alignment orientation, and attached to and proliferated on the acellular kidney sections, maintaining their phenotype. The hiPSC-ECs were able to self-organize into chimeric kidney organoids to form vessel-like structures. Ex vivo infusion of hiPSC-ECs through the renal artery and vein of acellular kidneys resulted in the uniform distribution of the cells in all the vasculature compartments, from glomerular capillaries to peritubular capillaries and small vessels. Ultrastructural analysis of repopulated scaffolds through transmission and scanning electron microscopy demonstrated the presence of continuously distributed cells along the vessel wall, which was also confirmed by 3D reconstruction of z-stack images showing the continuity of endothelial cell coverage inside the vessels. Notably, the detection of fenestrae in the endothelium of glomerular capillaries but not in the vascular capillaries was clear evidence of site-specific endothelial cell specialisation.
Asunto(s)
Riñón/química , Neovascularización Fisiológica/genética , Organoides/crecimiento & desarrollo , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Vasos Sanguíneos/química , Vasos Sanguíneos/crecimiento & desarrollo , Diferenciación Celular/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio/química , Endotelio/crecimiento & desarrollo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/crecimiento & desarrollo , Organoides/química , RatasRESUMEN
A number of derivatives of 4-amino-6-hydroxy-2-mercaptopyrimidine ( 5) were synthesized and biologically evaluated as A 3 adenosine receptor (A 3 AR) antagonists. The new compounds were designed as open chain analogues of a triazolopyrimidinone derivative displaying submicromolar affinity for the A 3 AR, which had been previously identified using a 3D database search. Substituents R, R', and R'' attached to the parent compound 5 were chosen according to factorial design and stepwise lead optimization approaches, taking into account the essentially hydrophobic nature of the A 3 AR binding site. As a result, 5m (R = n-C 3H 7, R' = 4-ClC 6H 4CH 2, R'' = CH 3) was identified among the pyrimidine derivatives as the ligand featuring the best combination of potency and selectivity for the target receptor. This compound binds to the A 3 AR with a K i of 3.5 nM and is devoid of appreciable affinity for the A 1, A 2A, and A 2B ARs.
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Antagonistas del Receptor de Adenosina A3 , Pirimidinas/farmacología , Compuestos de Sulfhidrilo/farmacología , Antagonistas del Receptor de Adenosina A1 , Antagonistas del Receptor de Adenosina A2 , Animales , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Células CHO , Simulación por Computador , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Estereoisomerismo , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/químicaRESUMEN
The study of some 4-substituted-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, designed as hA(3) adenosine receptor antagonists, is reported. The new compounds bear on the four-position different acylamino, sulfonylamino, benzylureido and benzyloxy moieties, which have also been combined with a para-methoxy group on the 2-phenyl ring or with a nitro residue at the six-position. Many derivatives show high hA(3) adenosine receptor affinities and selectivities both versus hA(1) and hA(2A) receptors. The observed structure-affinity relationships of this class of antagonists have been exhaustively rationalized using the recently published ligand-based homology modeling (LBHM) approach. The selected 4-bismethanesulfonylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (13), which shows high hA(3) affinity (K(i)=5.5nM) and selectivity versus hA(1), hA(2A) (both selectivity ratios>1800) and hA(2B) (cAMP assay, IC(50)>10,000nM) receptors, was tested in an in vitro rat model of cerebral ischemia, proving to be effective in preventing the failure of synaptic activity, induced by oxygen and glucose deprivation in the hippocampus, and in delaying the occurrence of anoxic depolarization.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Modelos Moleculares , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Animales , Unión Competitiva , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Isquemia Encefálica/prevención & control , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Humanos , Enlace de Hidrógeno , Ligandos , Unión Proteica , Quinoxalinas/metabolismo , Ratas , Receptor de Adenosina A3/metabolismo , Receptor de Adenosina A3/fisiología , Rodopsina/química , Homología Estructural de Proteína , Relación Estructura-Actividad , Triazoles/metabolismo , Xantinas/metabolismo , Xantinas/farmacologíaRESUMEN
Focal Segmental Glomerulosclerosis (FSGS) is the typical renal histologic lesion in familial steroid-resistant nephrotic syndrome, for which there is currently no treatment. Dysfunction of the glomerular podocyte, a specialized cell that forms the glomerular filtration barrier, is central in the pathogenesis of FSGS. Here, we reported the generation of two isogenic iPS cell lines from a patient affected by FSGS, carrying the c.565Gâ¯>â¯A mutation in the PAX2 gene. The iPS cell lines we generated expressed pluripotency markers at the mRNA and protein levels and differentiated into all three germ layers. These iPSCs will be instrumental in understanding FSGS pathogenesis.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/genética , Células Madre Pluripotentes Inducidas/metabolismo , Factor de Transcripción PAX2/genética , Heterocigoto , Humanos , Masculino , MutaciónRESUMEN
The lack of engineering systems able to faithfully reproduce complex kidney structures in vitro has made it difficult to efficiently model kidney diseases and development. Using polydimethylsiloxane (PDMS) scaffolds and a kidney-derived cell line we developed a system to rapidly engineer custom-made 3D tubules with typical renal epithelial properties. This system was successfully employed to engineer patient-specific tubules, to model polycystic kidney disease (PKD) and test drug efficacy, and to identify a potential new pharmacological treatment. By optimizing our system we constructed functional ureteric bud (UB)-like tubules from human induced pluripotent stem cells (iPSCs), and identified a combination of growth factors that induces budding morphogenesis like embryonic kidneys do. Finally, we applied this assay to investigate budding defects in UB-like tubules derived from a patient with a PAX2 mutation. Our system enables the modeling of human kidney disease and development, drug testing and discovery, and lays the groundwork for engineering anatomically correct kidney tissues in vitro and developing personalized medicine applications.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Túbulos Renales/citología , Técnicas de Cultivo de Órganos/métodos , Factor de Transcripción PAX2/genética , Enfermedades Renales Poliquísticas/patología , Animales , Diferenciación Celular , Perros , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Células de Riñón Canino Madin Darby , Modelos Biológicos , Mutación , Enfermedades Renales Poliquísticas/genética , Medicina de Precisión , Andamios del TejidoRESUMEN
This paper reports the study of some 2-arylpyrazolo[3,4-c]quinolin-4-ones, 4-amines, and 4-amino-substituted derivatives designed as human A3 adenosine receptor (AR) antagonists. Most of the herein reported compounds showed a nanomolar affinity toward the hA3 receptor subtype and different degrees of selectivity that resulted to be strictly dependent on the presence and nature of the substituent on the 4-amino group. Bulky and lipophilic acyl groups, as well as the benzylcarbamoyl residue, afforded highly potent and selective hA3 receptor antagonists. The selected 4-diphenylacetylamino-2-phenylpyrazoloquinoline (25) and 4-dibenzoylamino-2-(4-methoxyphenyl)pyrazoloquinoline (36), tested in an in vitro rat model of cerebral ischemia, prevented the irreversible failure of synaptic activity induced by oxygen and glucose deprivation in the hippocampus. The observed structure-affinity relationships of this class of antagonists were also exhaustively rationalized using the recently published ligand-based homology modeling (LBHM) approach.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Benzamidas/síntesis química , Modelos Moleculares , Pirazoles/síntesis química , Quinolinas/síntesis química , Animales , Benzamidas/química , Benzamidas/farmacología , Isquemia Encefálica/fisiopatología , Células CHO , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/biosíntesis , Electrofisiología , Hipocampo/fisiopatología , Humanos , Técnicas In Vitro , Ligandos , Masculino , Pirazoles/química , Pirazoles/farmacología , Quinolinas/química , Quinolinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Relación Estructura-Actividad , Transmisión Sináptica , Testículo/metabolismoRESUMEN
Binding assays on human A1, A2A, and A3 adenosine receptors (ARs) and functional studies on A2B ARs revealed that various 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones VIII, previously reported as ligands at the central benzodiazepine receptor (BzR), possess nanomolar affinity at the A3 AR. Replacement of the amide of VIII with an amidine moiety gave the 5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-ones IX, which maintain a nanomolar potency at the A3 AR with selectivity over the BzR. Insertion of a p-methoxybenzoyl at the 5-amino moiety enhanced A3 AR affinity and selectivity over the A1, A2A, and A2B ARs. The best result of our lead optimization efforts is 9-chloro-5-(4-methoxybenzoyl)amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one (23), which displayed a Ki of 1.6 nM at the A3 AR and no significant affinity at the other ARs or the BzR. Docking simulations on selected ligands into a model of the A3 AR allowed us to rationalize the structure-activity relationships of phenyltriazolobenzotriazindiones VIII and aminophenyltriazolobenzotriazinones IX at the molecular level.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Triazinas/síntesis química , Triazoles/síntesis química , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Triazinas/química , Triazinas/farmacología , Triazoles/química , Triazoles/farmacologíaRESUMEN
A structural investigation on some 4-amido-2-phenyl-1,2-dihydro-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, designed as human A3 adenosine receptor (hA3 AR) antagonists, is described. In the new derivatives, some acyl residues with different steric bulk were introduced on the 4-amino group, and their combination with the 4-methoxy group on the 2-phenyl moiety, and/or the 6-nitro/6-amino substituent on the fused benzo ring, was also evaluated. Most of the new derivatives were potent and selective hA3 AR antagonists. SAR analysis showed that hindering and lipophilic acyl moieties not only are well tolerated but even ameliorate the hA3 affinity. Interestingly, the 4-methoxy substituent on the appended 2-phenyl moiety, as well as the 6-amino group, always exerted a positive effect, shifting the affinity toward the hA3 receptor subtype. In contrast, the 6-nitro substituent exerted a variable effect. An intensive molecular modeling investigation was performed to rationalize the experimental SAR findings.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Modelos Moleculares , Quinoxalinas/síntesis química , Triazoles/síntesis química , Animales , Unión Competitiva , Células CHO , Bovinos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Ligandos , Membranas , Quinoxalinas/química , Quinoxalinas/farmacología , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacologíaRESUMEN
Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Podocitos/citología , Actinina/genética , Actinina/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Diferenciación Celular , Células Cultivadas , Cuerpos Embrioides/metabolismo , Cuerpos Embrioides/fisiología , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Cariotipo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Podocitos/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMEN
Acute kidney injury (AKI) is one of the most relevant health issues, leading to millions of deaths. The magnitude of the phenomenon remarks the urgent need for innovative and effective therapeutic approaches. Cell-based therapy with renal progenitor cells (RPCs) has been proposed as a possible strategy. Studies have shown the feasibility of directing embryonic stem cells or induced Pluripotent Stem Cells (iPSCs) towards nephrogenic intermediate mesoderm and metanephric mesenchyme (MM). However, the functional activity of iPSC-derived RPCs has not been tested in animal models of kidney disease. Here, through an efficient inductive protocol, we directed human iPSCs towards RPCs that robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin-mice with AKI. These results demonstrate that iPSCs are a valuable source of engraftable cells with regenerative activity for kidney disease and create the basis for future applications in stem cell-based therapy.
Asunto(s)
Lesión Renal Aguda/terapia , Células Madre Pluripotentes Inducidas/citología , Riñón/citología , Trasplante de Células Madre , Células Madre/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
The P2Y-like receptor GPR17 has been reported to respond to both uracil nucleotides and cysteinyl-leukotrienes (cysLTs), such as UDP-glucose and LTD(4). Our previous data suggest a potential role for GPR17 in regulation of both cell viability and differentiation state of central nervous system cells. On this basis, in the present paper we investigated the effect of GPR17 receptor ligands on PC12 cell viability, following induction of morphological differentiation by nerve growth factor (NGF). In addition, the role of GPR17 ligands, either alone or in combination with growth factors, on the degree of PC12 cell differentiation was investigated. GPR17, which was not basally expressed in undifferentiated PC12 cells, was specifically induced by a 10day-treatment with NGF, suggesting a role in the control of neuronal specification. Both UDP-glucose and LTD(4), agonists at the nucleotide and cysLT GPR17 binding sites, respectively, induced a significant pro-survival effect on PC12 cells after priming with NGF. By in vitro silencing experiments with specific small interfering RNAs and by using receptor antagonists, we confirmed that the agonist effects are indeed mediated by the selective activation of GPR17. We also demonstrated that GPR17 agonists act, both alone and synergistically with NGF, to promote neurite outgrowth in PC12 cells. In addition, GPR17 ligands were able to confer an NGF-like activity to the epidermal growth factor (EGF), that, under these experimental conditions, also promoted cell differentiation and neurite elongation. Finally, we show that GPR17 ligands activate the intracellular phosphorylation of both ERK 1/2 and p38 kinases, that have been identified as important signalling pathways for neurotrophins in PC12 cells. Our results establish GPR17 as a neurotrophic regulator for neuronal-like cells and suggest a possible interplay between endogenous uracil derivatives, cysLTs and NGF in the signalling pathways involved in neuronal survival and differentiation. They also represent the first direct demonstration, in a native system, that GPR17 can indeed be activated by uracil nucleotides and cysLTs, in line with what previously demonstrated in recombinant expression systems.
Asunto(s)
Supervivencia Celular , Neurogénesis , Neuronas/citología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Regulación de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Células PC12 , Interferencia de ARN , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Uracilo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Adenosine induces glioma cell proliferation by means of an antiapoptotic effect, which is blocked by cotreatment with selective A(3) AR antagonists. In this study, a novel series of N(2)-substituted pyrazolo[3,4-d]pyrimidines 2a-u was developed as highly potent and selective A(3) AR antagonists. The most performing compounds were derivatives 2a (R(1) = CH(3) and R(2) = COC(6)H(5); K(i) 334, 728, and 0.60 nM at the human A(1), A(2A), and A(3) ARs, respectively) and 2b (R(1) = CH(3) and R(2) = COC(6)H(4)-4-OCH(3); K(i) 1037, 3179, and 0.18 nM at the human A(1), A(2A), and A(3) ARs, respectively), which counteracted the effect of the A(3) AR agonists Cl-IB-MECA and IB-MECA on human glioma U87MG cell proliferation. This effect was concentration-dependent, with IC(50) values comparable to A(3) AR binding affinity values of 2a and 2b, thereby suggesting that their effects were receptor-mediated. Furthermore, the antiproliferative activity of the new compounds was demonstrated to be mediated by the block of A(3) AR agonist activation of intracellular kinases ERK 1/2.