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1.
Blood Adv ; 7(15): 3793-3805, 2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-37146271

RESUMO

The erythroid growth factor erythropoietin (EPO) is mainly produced by the kidneys in adult mammals and induces expansion of erythroid cells and iron use for hemoglobin synthesis. The liver also produces EPO at a lower level than the kidneys. Renal and hepatic EPO production is fundamentally regulated by hypoxia-inducible transcription factors (HIFs) in a hypoxia/anemia-inducible manner. Recently, small compounds that activate HIFs and EPO production in the kidneys by inhibiting HIF-prolyl hydroxylases (HIF-PHIs) have been launched to treat EPO-deficiency anemia in patients with kidney disease. However, the roles of the liver in the HIF-PHI-mediated induction of erythropoiesis and iron mobilization remain controversial. Here, to elucidate the liver contributions to the therapeutic effects of HIF-PHIs, genetically modified mouse lines lacking renal EPO-production ability were analyzed. In the mutant mice, HIF-PHI administration marginally increased plasma EPO concentrations and peripheral erythrocytes by inducing hepatic EPO production. The effects of HIF-PHIs on the mobilization of stored iron and on the suppression of hepatic hepcidin, an inhibitory molecule for iron release from iron-storage cells, were not observed in the mutant mice. These findings demonstrate that adequate induction of EPO mainly in the kidney is essential for achieving the full therapeutic effects of HIF-PHIs, which include hepcidin suppression. The data also show that HIF-PHIs directly induce the expression of duodenal genes related to dietary iron intake. Furthermore, hepatic EPO induction is considered to partially contribute to the erythropoietic effects of HIF-PHIs but to be insufficient to compensate for the abundant EPO induction by the kidneys.


Assuntos
Anemia , Eritropoetina , Camundongos , Animais , Eritropoese , Hepcidinas/genética , Preparações Farmacêuticas , Eritropoetina/farmacologia , Eritropoetina/genética , Eritropoetina/metabolismo , Rim , Anemia/tratamento farmacológico , Ferro/metabolismo , Hipóxia/metabolismo , Mamíferos/metabolismo
2.
Biochem Pharmacol ; 197: 114939, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35114188

RESUMO

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glicina/análogos & derivados , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Membranas Intracelulares/metabolismo , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Esterificação/efeitos dos fármacos , Esterificação/fisiologia , Glicina/metabolismo , Glicina/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fatores de Tempo , Resultado do Tratamento , Células Tumorais Cultivadas
3.
Kidney Int ; 101(1): 92-105, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34767829

RESUMO

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.


Assuntos
Metabolismo dos Lipídeos , Voo Espacial , Animais , Pressão Sanguínea/genética , Calcificação Fisiológica , Expressão Gênica , Rim/metabolismo , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo
4.
STAR Protoc ; 2(4): 100826, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34585160

RESUMO

Renal interstitial fibroblasts are responsible for producing the erythroid growth factor Epo and the vasopressor renin in addition to kidney fibrosis, in which they are transformed into myofibroblasts. Therefore, analyses of fibroblasts may elucidate the complex mechanisms of kidney diseases. However, the fragility of these cells makes their isolation for in vitro analyses and ex vivo cultivation difficult. We have overcome these difficulties by mildly dissociating mouse kidneys and coculturing fibroblasts with other kidney cells in semisolid medium. For complete details on the use and execution of this protocol, please refer to Sato et al. (2019a) and Miyauchi et al. (2021).


Assuntos
Fibroblastos , Nefropatias , Animais , Fibroblastos/metabolismo , Fibrose , Rim/metabolismo , Nefropatias/metabolismo , Camundongos , Miofibroblastos/metabolismo
5.
Mol Biol Cell ; 31(8): 741-752, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32049581

RESUMO

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.


Assuntos
Movimento Celular/fisiologia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Amidas/farmacologia , Animais , Polaridade Celular , Colágeno , Citoesqueleto/fisiologia , Desmossomos/fisiologia , Cães , Géis , Técnicas de Silenciamento de Genes , Queratina-18/antagonistas & inibidores , Queratina-18/genética , Queratina-18/fisiologia , Queratina-8/antagonistas & inibidores , Queratina-8/genética , Queratina-8/fisiologia , Células Madin Darby de Rim Canino , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Estresse Mecânico , Imagem com Lapso de Tempo , gama Catenina/antagonistas & inibidores , gama Catenina/genética , gama Catenina/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/fisiologia
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