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Diabetes ; 67(10): 2096-2106, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30065034


Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide, but its molecular pathogenesis is not well defined, and there are no specific treatments. In humans, there is a strong genetic component determining susceptibility to DN. However, specific genes controlling DN susceptibility in humans have not been identified. In this study, we describe a mouse model combining type 1 diabetes with activation of the renin-angiotensin system (RAS), which develops robust kidney disease with features resembling human DN: heavy albuminuria, hypertension, and glomerulosclerosis. Additionally, there is a powerful effect of genetic background regulating susceptibility to nephropathy; the 129 strain is susceptible to kidney disease, whereas the C57BL/6 strain is resistant. To examine the molecular basis of this differential susceptibility, we analyzed the glomerular transcriptome of young mice early in the course of their disease. We find dramatic differences in regulation of immune and inflammatory pathways, with upregulation of proinflammatory pathways in the susceptible (129) strain and coordinate downregulation in the resistant (C57BL/6) strain. Many of these pathways are also upregulated in rat models and in humans with DN. Our studies suggest that genes controlling inflammatory responses, triggered by hyperglycemia and RAS activation, may be critical early determinants of susceptibility to DN.

Nefropatias Diabéticas/genética , Nefropatias Diabéticas/imunologia , Inflamação/genética , Inflamação/imunologia , Nefropatias/genética , Nefropatias/imunologia , Animais , Glicemia/genética , Glicemia/imunologia , Western Blotting , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
Sci Rep ; 8(1): 2818, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29434311


Shortage of functional hepatocytes hampers drug safety testing and therapeutic applications because mature hepatocytes cannot be expanded and maintain functions in vitro. Recent studies have reported that liver progenitor cells can originate from mature hepatocytes in vivo. Derivation of proliferating progenitor cells from mature hepatocytes, and re-differentiation into functional hepatocytes in vitro has not been successful. Here we report the derivation of novel mesenchymal-like stem cells (arHMSCs) from adult rat hepatocytes. Immunofluorescence and flow cytometry characterization of arHMSCs found expression of mesenchymal markers CD29, CD44, CD90, vimentin and alpha smooth muscle actin. These arHMSCs proliferated in vitro for 4 passages yielding 104 fold increase in cell number in 28 days, and differentiated into hepatocyte-like cells (arHMSC-H). The arHMSC-H expressed significantly higher level of hepatocyte-specific markers (200 fold for albumin and 6 fold for Cyp450 enzymes) than arHMSCs. The arHMSC-H also demonstrated dose response curves similar to primary hepatocytes for 3 of the 6 paradigm hepatotoxicants tested, demonstrating utility in drug safety testing applications.

Técnicas de Cultura de Células/métodos , Hepatócitos/metabolismo , Fígado/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Ratos , Ratos Wistar , Células-Tronco/citologia
Toxicol In Vitro ; 50: 47-53, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29366910


Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFß1 production in the co-cultures. Addition of 100 pg/ml TGFß1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFß1 as one of the important factors impairing drug induced CYP induction in co-cultures and suggests that caution needs to be exercised in the use of hepatocyte-fibroblast co-cultures for CYP induction studies.

Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Fibroblastos/metabolismo , Hepatócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Masculino , Camundongos , Células NIH 3T3 , Ratos Wistar
J Appl Toxicol ; 36(2): 320-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26201057


Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

Células Cultivadas/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação de Medicamentos/métodos , Interações Medicamentosas/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos
Hum Gene Ther ; 24(5): 508-19, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23527815


Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-ß1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

Técnicas de Transferência de Genes , Células Estreladas do Fígado , Fator de Crescimento de Hepatócito/uso terapêutico , Cirrose Hepática/genética , Cirrose Hepática/terapia , Animais , Ductos Biliares , Dimetilnitrosamina/toxicidade , Regulação da Expressão Gênica , Terapia Genética , Fator de Crescimento de Hepatócito/genética , Humanos , Lipossomos/uso terapêutico , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Ratos , Transgenes
Biomaterials ; 33(7): 2165-76, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22189144


Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 µm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.

Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/citologia , Modelos Biológicos , Esferoides Celulares/fisiologia , Animais , Células Cultivadas , Colágeno/metabolismo , Hepatócitos/fisiologia , Humanos , Masculino , Ratos , Ratos Wistar , Esferoides Celulares/citologia