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1.
Biomed Pharmacother ; 177: 116923, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38936192

RESUMO

Acute kidney injury (AKI), characterized by a sudden decline in kidney function involving tubular damage and epithelial cell death, can lead to progressive tissue fibrosis and chronic kidney disease due to interstitial fibroblast activation and tissue repair failures that lack direct treatments. After an AKI episode, surviving renal tubular cells undergo cycles of dedifferentiation, proliferation and redifferentiation while fibroblast activity increases and then declines to avoid an exaggerated extracellular matrix deposition. Appropriate tissue recovery versus pathogenic fibrotic progression depends on fine-tuning all these processes. Identifying endogenous factors able to affect any of them may offer new therapeutic opportunities to improve AKI outcomes. Galectin-8 (Gal-8) is an endogenous carbohydrate-binding protein that is secreted through an unconventional mechanism, binds to glycosylated proteins at the cell surface and modifies various cellular activities, including cell proliferation and survival against stress conditions. Here, using a mouse model of AKI induced by folic acid, we show that pre-treatment with Gal-8 protects against cell death, promotes epithelial cell redifferentiation and improves renal function. In addition, Gal-8 decreases fibroblast activation, resulting in less expression of fibrotic genes. Gal-8 added after AKI induction is also effective in maintaining renal function against damage, improving epithelial cell survival. The ability to protect kidneys from injury during both pre- and post-treatments, coupled with its anti-fibrotic effect, highlights Gal-8 as an endogenous factor to be considered in therapeutic strategies aimed at improving renal function and mitigating chronic pathogenic progression.


Assuntos
Injúria Renal Aguda , Fibrose , Ácido Fólico , Galectinas , Animais , Masculino , Camundongos , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Ácido Fólico/farmacologia , Galectinas/metabolismo , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos Endogâmicos C57BL
2.
Front Cell Dev Biol ; 10: 833175, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36568977

RESUMO

Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin-Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

3.
Cancers (Basel) ; 13(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34298835

RESUMO

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

4.
Elife ; 102021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34155970

RESUMO

The COVID-19 pandemic will likely take years to control globally, and constant epidemic surveillance will be required to limit the spread of SARS-CoV-2, especially considering the emergence of new variants that could hamper the effect of vaccination efforts. We developed a simple and robust - Phone Screen Testing (PoST) - method to detect SARS-CoV-2-positive individuals by RT-PCR testing of smartphone screen swab samples. We show that 81.3-100% of individuals with high-viral-load SARS-CoV-2 nasopharyngeal-positive samples also test positive for PoST, suggesting this method is effective in identifying COVID-19 contagious individuals. Furthermore, we successfully identified polymorphisms associated with SARS-CoV-2 Alpha, Beta, and Gamma variants, in SARS-CoV-2-positive PoST samples. Overall, we report that PoST is a new non-invasive, cost-effective, and easy-to-implement smartphone-based smart alternative for SARS-CoV-2 testing, which could help to contain COVID-19 outbreaks and identification of variants of concern in the years to come.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19 , Pandemias , SARS-CoV-2/genética , Smartphone , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/genética , Humanos
5.
Cell Rep ; 25(11): 3110-3122.e6, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30540943

RESUMO

Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8-a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins-is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Galectinas/metabolismo , Sinapses Imunológicas/metabolismo , Animais , Linfócitos B/citologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Galinhas , Linfonodos/metabolismo , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteólise , Ratos , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/citologia
6.
Sci Rep ; 8(1): 728, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335424

RESUMO

Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.


Assuntos
Fenômenos Químicos , DNA/metabolismo , Nanodiamantes/química , Silanos/metabolismo , Difusão Dinâmica da Luz , Microscopia de Força Atômica , Solubilidade , Sonicação
7.
Am J Physiol Renal Physiol ; 310(9): F812-20, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26841823

RESUMO

The majority of patients with obesity, insulin resistance, and metabolic syndrome have hypertension, but the mechanisms of hypertension are poorly understood. In these patients, impaired sodium excretion is critical for the genesis of Na(+)-sensitive hypertension, and prior studies have proposed a role for the epithelial Na(+) channel (ENaC) in this syndrome. We characterized high fat-fed mice as a model in which to study the contribution of ENaC-mediated Na(+) reabsorption in obesity and insulin resistance. High fat-fed mice demonstrated impaired Na(+) excretion and elevated blood pressure, which was significantly higher on a high-Na(+) diet compared with low fat-fed control mice. However, high fat-fed mice had no increase in ENaC activity as measured by Na(+) transport across microperfused cortical collecting ducts, electrolyte excretion, or blood pressure. In addition, we found no difference in endogenous urinary aldosterone excretion between groups on a normal or high-Na(+) diet. High fat-fed mice provide a model of metabolic syndrome, recapitulating obesity, insulin resistance, impaired natriuresis, and a Na(+)-sensitive elevation in blood pressure. Surprisingly, in contrast to previous studies, our data demonstrate that high fat feeding of mice impairs natriuresis and produces elevated blood pressure that is independent of ENaC activity and likely caused by increased Na(+) reabsorption upstream of the aldosterone-sensitive distal nephron.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Canais Epiteliais de Sódio/metabolismo , Resistência à Insulina , Obesidade/metabolismo , Sódio/farmacologia , Aldosterona/urina , Animais , Ritmo Circadiano , Dieta Hiperlipídica , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Natriurese , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Obesidade/etiologia , Sódio/urina , Sódio na Dieta/efeitos adversos
8.
Am J Physiol Renal Physiol ; 308(11): F1306-15, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25810438

RESUMO

The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm(2)) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.


Assuntos
Aldosterona/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Distais/metabolismo , Néfrons/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Túbulos Renais Distais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Néfrons/efeitos dos fármacos , Potássio/metabolismo
9.
J Am Soc Nephrol ; 25(11): 2445-57, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24744440

RESUMO

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


Assuntos
Aldosterona/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , MicroRNAs/metabolismo , Sódio/metabolismo , Aldosterona/genética , Animais , Anquirinas/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Canais Epiteliais de Sódio/metabolismo , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Luciferases/genética , Camundongos Endogâmicos C57BL , Néfrons/citologia , Néfrons/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais/fisiologia
10.
EMBO J ; 24(7): 1397-405, 2005 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-15775969

RESUMO

The BMP4 signaling pathway plays key roles during early embryonic development and for maintenance of adult homeostasis. In the extracellular space, BMP4 activity is regulated by a group of interacting molecules including the BMP antagonist Chordin, the metalloproteinase Tolloid and Twisted gastrulation (Tsg). In this study, we identified Biglycan (Bgn), a member of the small leucine-rich proteoglycan family, as a new extracellular modulator of BMP4 signaling. Xenopus Bgn (xBgn) is expressed uniformly in the ectoderm and mesoderm and their derivatives during development. Microinjection of Bgn mRNA induced secondary axes, dorsalized the mesoderm and inhibited BMP4 activity in Xenopus embryos. Biochemical experiments showed that Bgn binds BMP4 and Chordin, interaction that increased binding of BMP4 to Chordin. Bgn was also able to improve the efficiency of Chordin-Tsg complexes to block BMP4 activity. Using antisense morpholinos, we demonstrated that Bgn required Chordin to induce double axes in Xenopus. This work unveiled a new function for Bgn, its ability to regulate BMP4 signaling through modulation of Chordin anti-BMP4 activity.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais/fisiologia , Xenopus/embriologia , Animais , Biglicano , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/fisiologia , Primers do DNA , Ectoderma/metabolismo , Proteínas da Matriz Extracelular , Glicoproteínas/fisiologia , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mesoderma/metabolismo , Microinjeções , Oligonucleotídeos , Proteoglicanas/fisiologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Xenopus
11.
J Cell Sci ; 117(Pt 21): 5179-90, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15456854

RESUMO

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.


Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Sistema Nervoso/metabolismo , Proteoglicanas/genética , Proteoglicanas/fisiologia , Vulva/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Caenorhabditis elegans , Feminino , Deleção de Genes , Glicosilação , Proteoglicanas de Heparan Sulfato/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fenótipo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Sindecanas
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