RESUMO
The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.
Assuntos
Proteína Forkhead Box O3/metabolismo , Túbulos Renais Proximais/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Proteína Forkhead Box O3/genética , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , beta Catenina/genéticaRESUMO
New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1ß, HNF3ß [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPß and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Camundongos , Especificidade de ÓrgãosRESUMO
While hepatic cell lines are mainly used for in vitro drug induced toxicity studies, they exhibit limited functionalities. To overcome this, the authors have employed genetically engineered mouse hepatoma cells, Hepa/8F5, wherein expression of liver enriched transcription factors is induced by doxycycline leading to increased functionality. Further enhancement in functionality is achieved by spheroid culture in a previously developed 3D cell culture platform. Cells are seeded in presence of temperature-responsive poly(N-isopropylacrylamide) on poly(N-isopropylacrylamide--co-gelatin) cryogel scaffold based high throughput platform. Cells seeded in presence of poly(N-isopropylacrylamide) and induced with doxycycline exhibited highest functionalities. There is an increase of ≈26, 36, and 39% in albumin secretion, ammonia removal, and CYP3A4 activity, respectively. Morphological analysis showed arrest in cell proliferation and enlarged nucleus in presence of doxycyline and spheroid formation in presence of poly(N-isopropylacrylamide). Drug induced liver toxicity studies revealed that cells induced with doxycycline are resistive to tamoxifen but sensitive to acetaminophen whereas, cultures initiated in presence of poly(N-isopropylacrylamide) are resistive to both the drugs which is indicative of diffusional barrier of the spheroids. The authors conclude that Hepa/8F5 cells show enhanced functionality in cryogel based spheroid culture platform which can be successfully used for high throughput screening of hepatic toxicity in vitro.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Acrilamidas/farmacologia , Resinas Acrílicas/farmacologia , Albuminas/metabolismo , Amônia/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Camundongos , Polímeros/farmacologia , Temperatura , Fatores de TranscriçãoRESUMO
Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research.