RESUMO
The extracellular matrix (ECM) of tumors is a complex mix of components characteristic of the tissue of origin. In the majority of clear cell renal cell carcinomas (ccRCCs), the tumor suppressor VHL is inactivated. VHL controls matrix organization and its loss promotes a loosely organized and angiogenic matrix, predicted to be an early step in tumor formation. During tumor evolution, cancer-associated fibroblasts (CAFs) accumulate, and they are predicted to produce abundant ECM. The ccRCC ECM composition qualitatively resembles that of the healthy kidney cortex in which the tumor arises, but there are important differences. One is the quantitative difference between a healthy cortex ECM and a tumor ECM; a tumor ECM contains a higher proportion of interstitial matrix components and a lower proportion of basement membrane components. Another is the breakdown of tissue compartments in the tumor with mixing of ECM components that are physically separated in healthy kidney cortex. Numerous studies reviewed in this work reveal effects of specific ECM components on the growth and invasive behaviors of ccRCCs, and extrapolation from other work suggests an important role for ECM in controlling ccRCC tumor rigidity, which is predicted to be a key determinant of invasive behavior.
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Clinical association studies suggest that FOXD1 is a determinant of patient outcome in clear cell renal cell carcinoma (ccRCC), and laboratory investigations have defined a role for this transcription factor in controlling the growth of tumors through regulation of the G2/M cell cycle transition. We hypothesized that the identification of pathways downstream of FOXD1 may define candidates for pharmacological modulation to suppress the G2/M transition in ccRCC. We developed an analysis pipeline that utilizes RNA sequencing, transcription factor binding site analysis, and phenotype validation to identify candidate effectors downstream from FOXD1. Compounds that modulate candidate pathways were tested for their ability to cause growth delay at G2/M. Three targets were identified: FOXM1, PME1, and TMEM167A, which were targeted by compounds FDI-6, AMZ-30, and silibinin, respectively. A 3D ccRCC tumor replica model was used to investigate the effects of these compounds on the growth of primary cells from five patients. While silibinin reduced 3D growth in a subset of tumor replicas, FDI-6 reduced growth in all. This study identifies tractable pathways to target G2/M transition and inhibit ccRCC growth, demonstrates the applicability of these strategies across patient tumor replicas, and provides a platform for individualized patient testing of compounds that inhibit tumor growth.
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Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.
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Development of a 3D, biomaterials-based model for clear cell renal cell carcinoma (ccRCC) would be advantageous for understanding disease progression in vitro. This study demonstrated the development of lyophilized silk scaffolds that mechanically match the experimentally determined Young's modulus for ex vivo ccRCC samples and normal kidney tissue. Scaffolds fabricated from silk solutions ranging from 3 to 12% (w/v) were evaluated through mechanical testing. Following mechanical characterization of ccRCC samples, it was demonstrated that 6% silk scaffolds mechanically matched ccRCC samples. No impact of pathological grade and stage on the calculated ccRCC modulus was observed and all tumors evaluated mechanically matched the 6% silk scaffold formulation. Stratifying tissue specimens based upon histological observations (e.g. evidence of high levels of collagen deposition) resulted in no significant differences between groups. To investigate the impact of a mechanically matched culturing environment on in vitro ccRCC disease characteristics a model ccRCC cell line, 786-O, was utilized. Scaffolded 786-O cells demonstrated increased lipid droplet accumulation, a hallmark of ccRCC, compared to standard two-dimensional (2D) culture conditions. Additionally, scaffolded 786-O cells demonstrated increased expression of genes associated with ccRCC aggressiveness (ex. VEGFA, TNF, and IL-6) or immune markers under investigation as therapeutic targets (ex. PDL1, CTLA4). Comparison with 786-O cells grown on non-mechanically matched scaffolds demonstrated that these improved ccRCC characteristics were driven by scaffold modulus. Overall, our findings support the use of silk scaffolds in replicating physiologic tumor behavior for clear cell renal cell carcinoma and provide a platform for investigating disease progression.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Materiais Biocompatíveis , Proliferação de Células , Colágeno , Humanos , Seda , Alicerces TeciduaisRESUMO
BACKGROUND: Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported. METHODS: Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database. RESULTS: FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation. CONCLUSIONS: We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.
Assuntos
Carcinoma de Células Renais/genética , Divisão Celular/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Técnicas de Inativação de Genes , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação/genética , RNA-Seq , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stem cell-derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long-term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide-ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including-but not limited to-the kidney.
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Linfonodos/crescimento & desenvolvimento , Modelos Biológicos , Néfrons/citologia , Néfrons/crescimento & desenvolvimento , Organogênese , Células-Tronco/citologia , Animais , Humanos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Silk fibroin biomaterials are highly versatile in terms of materials formation and functionalization, with applications in tissue engineering and drug delivery, but necessitate modifications for optimized biological activity. Herein, a facile, avidin-based technique is developed to noncovalently functionalize silk materials with bioactive molecules. The ability to adsorb avidin to silk surfaces and subsequently couple biotinylated macromolecules via avidin-biotin interaction is described. This method better preserved functionality than standard covalent coupling techniques using carbodiimide cross-linking chemistry. The controlled release of avidin from the silk surface was demonstrated by altering the adsorption parameters. Application of this technique to culturing human foreskin fibroblasts (hFFs) and human mesenchymal stem cells (hMSCs) on arginine-glycine-aspartic-acid-modified (RGD-modified) silk showed increased cell growth over a seven-day period. This technique provides a facile method for the versatile functionalization of silk materials for biomedical applications including tissue engineering, drug delivery, and biological sensing.
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Avidina/química , Fibroínas/química , Alicerces Teciduais/química , Adsorção , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Oligopeptídeos/química , Alicerces Teciduais/efeitos adversosRESUMO
Formation of a functional kidney depends on the balance between renewal and differentiation of nephron progenitors. Failure to sustain this balance can lead to kidney failure or stem cell tumors. For nearly 60 years, we have known that signals from an epithelial structure known as the ureteric bud were essential for maintaining this balance. More recently it was discovered that one molecule, Wnt9b, was necessary for both renewal and differentiation of the nephron progenitor cells. How one ligand signaling through one transcription factor promoted two seemingly contradictory cellular processes was unclear. In this study, we show that Wnt9b/beta-catenin signaling alone is sufficient to promote both renewal and differentiation. Moreover, we show that discrete levels of beta-catenin can promote these two disparate fates, with low levels fostering progenitor renewal and high levels driving differentiation. These results provide insight into how Wnt9b regulates distinct target genes that balance nephron progenitor renewal and differentiation.
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Néfrons/fisiologia , beta Catenina/metabolismo , beta Catenina/fisiologia , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Néfrons/embriologia , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
The final nephron number in an adult kidney is regulated by nephron progenitor cell availability and collecting duct branching in the fetal period. Fetal environmental perturbations that cause reductions in cell numbers in these two compartments result in low nephron endowment. Previous work has shown that maternal dietary factors influence nephron progenitor cell availability, with both caloric restriction and protein deprivation leading to reduced cell numbers through apoptosis. In this study, we evaluate the consequences of inducing nephron progenitor cell death on progenitor niche dynamics and on nephron endowment. Depletion of approximately 40% of nephron progenitor cells by expression of diphtheria toxin A at embryonic day 15 in the mouse results in 10-20% nephron reduction in the neonatal period. Analysis of cell numbers within the progenitor cell pool following induction of apoptosis reveals a compensatory response in which surviving progenitor cells increase their proliferation and replenish the niche. The proliferative response is temporally associated with infiltration of macrophages into the nephrogenic zone. Colony stimulating factor 1 (CSF1) has a mitogenic effect on nephron progenitor cells, providing a potential explanation for the compensatory proliferation. However, CSF1 also promotes interstitial cell proliferation, and the compensatory response is associated with interstitial expansion in recovering kidneys which can be pharmacologically inhibited by treatment with clodronate liposomes. Our findings suggest that the fetal kidney employs a macrophage-dependent compensatory regenerative mechanism to respond to acute injury caused by death of nephron progenitor cells, but that this regenerative response is associated with neonatal interstitial expansion.
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Néfrons/patologia , Células-Tronco/patologia , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Contagem de Células , Morte Celular , Proliferação de Células , Sobrevivência Celular , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Néfrons/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismoRESUMO
Evidence suggests that stromal cells play critical roles in tumor growth. Uncovering new mechanisms that control stromal cell behavior and their accumulation within tumors may lead to development of more effective treatments. We provide evidence that the HU177 cryptic collagen epitope is selectively generated within human ovarian carcinomas and this collagen epitope plays a role in SKOV-3 ovarian tumor growth in vivo. The ability of the HU177 epitope to regulate SKOV-3 tumor growth depends in part on its ability to modulate stromal cell behavior because targeting this epitope inhibited angiogenesis and, surprisingly, the accumulation of α-smooth muscle actin-expressing stromal cells. Integrin α10ß1 can serve as a receptor for the HU177 epitope in α-smooth muscle actin-expressing stromal cells and subsequently regulates Erk-dependent migration. These findings are consistent with a mechanism by which the generation of the HU177 collagen epitope provides a previously unrecognized α10ß1 ligand that selectively governs angiogenesis and the accumulation of stromal cells, which in turn secrete protumorigenic factors that contribute to ovarian tumor growth. Our findings provide a new mechanistic understanding into the roles by which the HU177 epitope regulates ovarian tumor growth and provide new insight into the clinical results from a phase 1 human clinical study of the monoclonal antibody D93/TRC093 in patients with advanced malignant tumors.
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Proliferação de Células , Colágeno/metabolismo , Epitopos , Neoplasias Ovarianas/patologia , Microambiente Tumoral/fisiologia , Animais , Western Blotting , Adesão Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno/química , Feminino , Xenoenxertos , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/metabolismoRESUMO
PURPOSE OF REVIEW: This article gives an overview of important new advances relating to kidney bioengineering. RECENT FINDINGS: Directed differentiation studies have shown that proximal tubules, distal tubules, podocytes, collecting ducts, interstitium and endothelial cells can be generated from patient-derived stem cells using standardized protocols. One caveat to the interpretation of these studies is that the physiological characteristics of differentiated cells remain to be defined. Another important area of progress is scaffolding. Both decellularized organs and polymeric materials are being used as platforms for three-dimensional growth of kidney tissue, and key distinctions between these approaches are discussed. SUMMARY: In the past 3 years, it has become clear that building kidney tissue is feasible. The laboratory-grown kidney is an attainable goal if efforts are focused on refining directed differentiation procedures to optimize cell function and on developing scaffolding strategies that ensure physiological function at the tissue level.
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Falência Renal Crônica/cirurgia , Transplante de Rim , Rim , Técnicas de Cultura de Órgãos , Células-Tronco , Engenharia Tecidual , Animais , Bioengenharia , Diferenciação Celular , Células Endoteliais , Humanos , Túbulos Renais , Túbulos Renais Coletores , Podócitos , Alicerces TeciduaisRESUMO
Renal dysplasia, the leading cause of renal failure in children, is characterized by disrupted branching of the collecting ducts and primitive tubules, with an expansion of the stroma, yet a role for the renal stroma in the genesis of renal dysplasia is not known. Here, we demonstrate that expression of ß-catenin, a key transcriptional co-activator in renal development, is markedly increased in the expanded stroma in human dysplastic tissue. To understand its contribution to the genesis of renal dysplasia, we generated a mouse model that overexpresses ß-catenin specifically in stromal progenitors, termed ß-cat(GOF-S) . Histopathological analysis of ß-cat(GOF) (-S) mice revealed a marked expansion of fibroblast cells surrounding primitive ducts and tubules, similar to defects observed in human dysplastic kidneys. Characterization of the renal stroma in ß-cat(GOF) (-S) mice revealed altered stromal cell differentiation in the expanded renal stroma demonstrating that this is not renal stroma but instead a population of stroma-like cells. These cells overexpress ectopic Wnt4 and Bmp4, factors necessary for endothelial cell migration and blood vessel formation. Characterization of the renal vasculature demonstrated disrupted endothelial cell migration, organization, and vascular morphogenesis in ß-cat(GOF) (-S) mice. Analysis of human dysplastic tissue demonstrated a remarkably similar phenotype to that observed in our mouse model, including altered stromal cell differentiation, ectopic Wnt4 expression in the stroma-like cells, and disrupted endothelial cell migration and vessel formation. Our findings demonstrate that the overexpression of ß-catenin in stromal cells is sufficient to cause renal dysplasia. Further, the pathogenesis of renal dysplasia is one of disrupted stromal differentiation and vascular morphogenesis. Taken together, this study demonstrates for the first time the contribution of stromal ß-catenin overexpression to the genesis of renal dysplasia. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Diferenciação Celular , Túbulos Renais Proximais/anormalidades , Anormalidades Urogenitais/genética , Remodelação Vascular , beta Catenina/genética , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Transgênicos , Fenótipo , Transdução de Sinais , Células Estromais/metabolismo , Anormalidades Urogenitais/metabolismo , Anormalidades Urogenitais/patologia , Proteína Wnt4/genética , Proteína Wnt4/metabolismo , beta Catenina/metabolismoRESUMO
The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31(+) cKit(-) endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.
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Endotélio/embriologia , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína Smad1/genética , Proteína Smad5/genética , Animais , Sequência de Bases , Linhagem Celular , Endotélio/metabolismo , Fator de Transcrição GATA2/genética , Técnicas de Silenciamento de Genes , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/genéticaRESUMO
Contrary to its classic role in restraining cell proliferation, we demonstrate here a divergent function of p53 in the maintenance of self-renewal of the nephron progenitor pool in the embryonic mouse kidney. Nephron endowment is regulated by progenitor availability and differentiation potential. Conditional deletion of p53 in nephron progenitor cells (Six2Cre(+);p53(fl/fl)) induces progressive depletion of Cited1(+)/Six2(+) self-renewing progenitors and loss of cap mesenchyme (CM) integrity. The Six2(p53-null) CM is disorganized, with interspersed stromal cells and an absence of a distinct CM-epithelia and CM-stroma interface. Impaired cell adhesion and epithelialization are indicated by decreased E-cadherin and NCAM expression and by ineffective differentiation in response to Wnt induction. The Six2Cre(+);p53(fl/fl) cap has 30% fewer Six2(GFP(+)) cells. Apoptotic index is unchanged, whereas proliferation index is significantly reduced in accordance with cell cycle analysis showing disproportionately fewer Six2Cre(+);p53(fl/fl) cells in the S and G2/M phases compared with Six2Cre(+);p53(+/+) cells. Mutant kidneys are hypoplastic with fewer generations of nascent nephrons. A significant increase in mean arterial pressure is observed in early adulthood in both germline and conditional Six2(p53-null) mice, linking p53-mediated defects in kidney development to hypertension. RNA-Seq analyses of FACS-isolated wild-type and Six2(GFP(+)) CM cells revealed that the top downregulated genes in Six2Cre(+);p53(fl/fl) CM belong to glucose metabolism and adhesion and/or migration pathways. Mutant cells exhibit a â¼ 50% decrease in ATP levels and a 30% decrease in levels of reactive oxygen species, indicating energy metabolism dysfunction. In summary, our data indicate a novel role for p53 in enabling the metabolic fitness and self-renewal of nephron progenitors.
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Néfrons/citologia , Néfrons/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Pressão Sanguínea , Adesão Celular/genética , Ciclo Celular , Movimento Celular/genética , Proliferação de Células , Senescência Celular/genética , Metabolismo Energético/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Células Germinativas/citologia , Proteínas de Homeodomínio/metabolismo , Integrases/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Organogênese/genética , Fenótipo , Células Estromais/citologia , Células Estromais/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in ß-catenin activity along the axis of the nephron tubule. By modifying ß-catenin activity, we force cells within nephrons to differentiate according to the imposed ß-catenin activity level, thereby causing spatial shifts in nephron segments. The ß-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises ß-catenin activity and promotes segment identities associated with low ß-catenin activity. ß-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating ß-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.
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Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Néfrons/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Receptores Notch/metabolismo , beta Catenina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Cultura em Câmaras de Difusão , Embrião de Mamíferos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Néfrons/citologia , Néfrons/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Transdução de Sinais , beta Catenina/genéticaRESUMO
BACKGROUND: DAPK2 is a pro-apoptotic protein kinase that associates with TGFß receptors. The homolog DAPK1 has been shown to mediate apoptosis in kidney injury. Expression databases indicate that DAPK2 is expressed in the kidney, and in this work we investigate the localization of renal DAPK2 expression and its role in the kidney. RESULTS: Immunostaining demonstrates DAPK2 expression in interstitial cells of the renal cortex including PDGFRß-positive pericytes and the CD73-positive erythropoietin-expressing fibroblast population. Tubulointerstitial fibrosis in experimental CKD arises directly from resident interstitial cells, and we therefore evaluated the expression of DAPK2 in the expanded interstitium of mice with kidney disease induced by chronic cisplatin administration. Expanded renal interstitium in these animals was negative for DAPK2 expression, but healthy areas of the kidney in which the tubular interstitium had not expanded expressed DAPK2 at levels similar to the uninjured control. Dapk2 null mice were generated to evaluate if DAPK2 is required for formation of the kidney, or its maintenance in the adult. Kidneys of Dapk2 null mice did not show overt malformations or age-related degeneration, but did show a slight increase in the number of interstitial fibroblasts. Differences were seen between Dapk2 null mice and wild type controls in the response to tubulointerstitial fibrosis caused by chronic cisplatin administration. Although mutant and wild type mice displayed comparable levels of alpha smooth muscle actin, interstitial proliferation and SMAD2 signaling, Dapk2 null mice showed reduced interstitial collagen accumulation. CONCLUSIONS: In the kidney, DAPK2 is strongly and specifically expressed in interstitial cells of the cortex, providing a useful marker for this important cell population. Dapk2 null mice are phenotypically normal under steady state conditions, but display some resistance to extracellular matrix deposition in experimental renal fibrosis indicating that DAPK2 plays a profibrotic role in kidney injury.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Rim/enzimologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Apoptose , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Proteínas Quinases Associadas com Morte Celular/genética , Rim/citologia , Rim/efeitos dos fármacos , Camundongos , Camundongos KnockoutRESUMO
Embryonic nephron progenitor cells are segregated in molecularly distinct compartments of unknown function. Our study reveals an integral role for bone morphogenetic protein-SMAD in promoting transition of progenitors from the primitive Cbp/p300-interacting transactivator 1 expressing (CITED1+) compartment to the uniquely sine oculis-related homeobox 2 expressing (SIX2-only) compartment where they become inducible by wingless-type mouse mammary tumor virus integration site family member (WNT)/ß-catenin signaling. Significantly, CITED1(+) cells are refractory to WNT/ß-catenin induction. We propose a model in which the primitive CITED1(+) compartment is refractory to induction by WNT9b/ß-catenin, ensuring maintenance of undifferentiated progenitor cells for future nephrogenesis. Bone morphogenetic protein 7-SMAD is then required for transition to a distinct compartment in which cells become inducible by WNT9b/ß-catenin, allowing them to progress toward epithelialization.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Néfrons/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Néfrons/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Células-Tronco/citologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O(2)) compared with normoxia (21% O(2)). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H(2)O(2)) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H(2)O(2)/SOD2 production on BMP2 upregulation. Chondrocytes produced more H(2)O(2) under hypoxia than normoxia. H(2)O(2) addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H(2)O(2) production and only partly through the HIF1 pathway.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Doença de Legg-Calve-Perthes/metabolismo , Regulação para Cima , Animais , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , Modelos Animais de Doenças , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Superóxido Dismutase/metabolismo , Suínos , Regulação para Cima/efeitos dos fármacosRESUMO
FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC), which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.
Assuntos
Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/fisiologia , Rim/lesões , Rim/fisiopatologia , Animais , Sequência de Bases , Crescimento Celular , Primers do DNA/genética , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Fator 1 de Crescimento de Fibroblastos/sangue , Fibrose , Rim/irrigação sanguínea , Rim/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neutrófilos/patologia , Tamanho do Órgão , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais , Taurina/farmacologia , Cicatrização/efeitos dos fármacos , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Endogenous Bone Morphogenetic Protein (BMP) signaling plays a significant role in the kidney's recovery from acute injury and exogenous administration of BMP7 has therapeutic potential in numerous rodent models of renal injury and disease. However, in the healthy kidney endogenous BMP7 ligand is vigorously counteracted by extracellular antagonists such as USAG1 and CHRDL1. Little is known about the degree of BMP signaling and the ligands driving it in the healthy adult kidney. In this study we characterize basal BMP signaling in the healthy tubular nephron, and show that BMP2 is expressed in proximal nephron epithelial cells. Comparative gene profiling of proximal tubule cell responses to BMP2 and BMP7 does not reveal any qualitative difference, suggesting that identical BMP gene targets may be activated in healthy and injured organs. Interestingly, our gene profiling analysis shows that BMP signaling activates a number of Notch regulated transcription factors, including HEY1. As in other biological systems, HEY1 functions as a negative feedback regulator of BMP2 expression in the proximal tubule. In summary, this work reveals endogenous BMP signaling patterns in the healthy human and mouse kidneys, and identifies novel gene targets, some of which are involved in the complex regulation of BMP signaling in the adult kidney.