RESUMO
The cellular and molecular mechanisms that underpin regeneration of the human lung are unknown, and the study of lung repair has been impeded by the necessity for reductionist models that may exclude key components. We hypothesized that multicellular epithelial and mesenchymal cell clusters or lung organoid units (LuOU) could be transplanted to recapitulate proximal and distal cellular structures of the native lung and airways. Transplantation of LuOU resulted in the growth of tissue-engineered lung (TELu) that contained the necessary cell types consistent with native adult lung tissue and demonstrated proliferative cells at 2 and 4 weeks. This technique recapitulated important elements of both mouse and human lungs featuring key components of both the proximal and distal lung regions. When LuOU were generated from whole lung, TELu contained key epithelial and mesenchymal cell types, and the origin of the cells was traced from both ActinGFP and SPCGFP donors to indicate that the cells in TELu were derived from the transplanted LuOU. Alveolar epithelial type 2 cells (AEC2s), club cells, ciliated cells marked by beta-tubulin IV, alveolar epithelial type I cells, Sox-2-positive proximal airway progenitors, p63-positive basal cells, and CGRP-positive pulmonary neuroendocrine cells were identified in the TELu. The mesenchymal components of peribronchial smooth muscle and nerve were identified with a CD31-positive donor endothelial cell contribution to TELu vasculature. TELu successfully grew from postnatal tissues from whole murine and human lung, distal murine lung, as well as murine and human trachea. These data support a model of postnatal lung regeneration containing the diverse cell types present in the entirety of the respiratory tract.
Assuntos
Linhagem da Célula , Proliferação de Células , Pulmão/citologia , Engenharia Tecidual/métodos , Traqueia/metabolismo , Cicatrização , Animais , Células Cultivadas , Humanos , Pulmão/fisiologia , Transplante de Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Traqueia/citologiaRESUMO
RATIONALE: Neonatal respiratory distress syndrome is a restrictive lung disease characterized by surfactant deficiency. Decreased vascular endothelial growth factor (VEGF), which demonstrates important roles in angiogenesis and vasculogenesis, has been implicated in the pathogenesis of restrictive lung diseases. Current animal models investigating VEGF in the etiology and outcomes of RDS require premature delivery, hypoxia, anatomically or temporally limited inhibition, or other supplemental interventions. Consequently, little is known about the isolated effects of chronic VEGF inhibition, started at birth, on subsequent developing lung structure and function. OBJECTIVES: To determine whether inducible, mesenchyme-specific VEGF inhibition in the neonatal mouse lung results in long-term modulation of AECII and whole lung function. METHODS: Triple transgenic mice expressing the soluble VEGF receptor sFlt-1 specifically in the mesenchyme (Dermo-1/rtTA/sFlt-1) were generated and compared to littermate controls at 3 months to determine the impact of neonatal downregulation of mesenchymal VEGF expression on lung structure, cell composition and function. Reduced tissue VEGF bioavailability has previously been demonstrated with this model. MEASUREMENTS AND MAIN RESULTS: Triple transgenic mice demonstrated restrictive lung pathology. No differences in gross vascular development or protein levels of vascular endothelial markers was noted, but there was a significant decrease in perivascular smooth muscle and type I collagen. Mutants had decreased expression levels of surfactant protein C and hypoxia inducible factor 1-alpha without a difference in number of type II pneumocytes. CONCLUSIONS: These data show that mesenchyme-specific inhibition of VEGF in neonatal mice results in late restrictive disease, making this transgenic mouse a novel model for future investigations on the consequences of neonatal RDS and potential interventions.
Assuntos
Pneumopatias/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Mesoderma/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Peso Corporal , Colágeno/química , Feminino , Regulação da Expressão Gênica , Hidroxiprolina/química , Modelos Lineares , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Testes de Função Respiratória , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Type 2 alveolar epithelial cells (AEC2) are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance. Depletion of this population is hypothesized to underlie various lung pathologies. Current models of lung injury rely on either uncontrolled, nonspecific destruction of alveolar epithelia or on targeted, nontitratable levels of fixed AEC2 ablation. We hypothesized that discrete levels of AEC2 ablation would trigger stereotypical and informative patterns of repair. To this end, we created a transgenic mouse model in which the surfactant protein-C promoter drives expression of a mutant SR39TK herpes simplex virus-1 thymidine kinase specifically in AEC2. Because of the sensitivity of SR39TK, low doses of ganciclovir can be administered to these animals to induce dose-dependent AEC2 depletion ranging from mild (50%) to lethal (82%) levels. We demonstrate that specific levels of AEC2 depletion cause altered expression patterns of apoptosis and repair proteins in surviving AEC2 as well as distinct changes in distal lung morphology, pulmonary function, collagen deposition, and expression of remodeling proteins in whole lung that persist for up to 60 days. We believe SPCTK mice demonstrate the utility of cell-specific expression of the SR39TK transgene for exerting fine control of target cell depletion. Our data demonstrate, for the first time, that specific levels of type 2 alveolar epithelial cell depletion produce characteristic injury repair outcomes. Most importantly, use of these mice will contribute to a better understanding of the role of AEC2 in the initiation of, and response to, lung injury.
Assuntos
Células Epiteliais Alveolares/patologia , Lesão Pulmonar/patologia , Fibrose Pulmonar/patologia , Regeneração , Células Epiteliais Alveolares/enzimologia , Animais , Apoptose , Proliferação de Células , Forma Celular , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ganciclovir/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Hiperóxia/complicações , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Lesão Pulmonar/fisiopatologia , Camundongos Transgênicos , Fenótipo , Regiões Promotoras Genéticas , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/fisiopatologia , Proteína C Associada a Surfactante Pulmonar/genética , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Fatores de Tempo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Congenital urinary tract obstruction is the single most important cause of childhood chronic kidney disease. We have previously demonstrated that human and primate fetal obstruction impairs the development, differentiation, and maturation of the kidney. Research using postnatal rodent models has primarily focused upon the role of proximal tubular injury, with few reports of collecting duct system pathology or the suitability of the postnatal models for examining injury to the distal nephron. We have employed the mouse unilateral ureteric obstruction (UUO) model and examined time points ranging from 1 to 14 days of obstruction. Many of the key features of fetal collecting duct injury are replicated in the postnatal mouse model of obstruction. Obstruction causes a sixfold increase in myofibroblast accumulation, two- to threefold dilatation of tubules of the distal nephron, 65% reduction of principal cell aquaporin 2 expression, 75% reduction of collecting duct intercalated cell abundance, and disruption of E-cadherin- and ßcatenin-mediated collecting duct epithelial adhesion. Notably, these features are shared by the distal and connecting tubules. This work confirms that distal nephron pathology is a significant component of postnatal mouse UUO. We have highlighted the utility of this model for investigating collecting duct and distal tubule injury and for identifying the underlying mechanisms of the distal nephron's contribution to the repair and fibrosis.
Assuntos
Néfrons/patologia , Obstrução Ureteral/patologia , Obstrução Ureteral/fisiopatologia , Análise de Variância , Animais , Apoptose/fisiologia , Caderinas/metabolismo , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Rim/química , Rim/metabolismo , Rim/patologia , Túbulos Renais Coletores/química , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/patologia , Cinética , Masculino , Camundongos , Néfrons/química , Néfrons/metabolismo , Estatísticas não Paramétricas , Obstrução Ureteral/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Obstructive nephropathy is a leading cause of CKD in children. The assessment of severity of renal impairment and the prediction of which children will progress to renal failure are, however, challenging. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This case-control study measured the urinary excretion of candidate biomarkers in 27 prevalent case-patients with posterior urethral valves (PUVs) and 20 age-matched controls, correlated their urinary concentration with GFR, and analyzed receiver-operating characteristic (ROC) curve and regression analyses to assess their performance as tests for low GFR. RESULTS: The median urinary protein-to-creatinine ratio was higher in children with PUV (45 g/mol; range, 5-361 g/mol) than in controls (7 g/mol; range, 3-43 g/mol) (P<0.01) and correlated inversely with renal function (r = -0.44; P<0.05). In whole urine, excretion of aquaporin-2 was significantly decreased, whereas that of TGFß and L1 cell adhesion molecule (L1CAM) was significantly increased. Whole-urine TGFß excretion correlated inversely with GFR (r = -0.53; P<0.05). As tests for low GFR, whole-urine TGFß, L1CAM, and urinary protein-to-creatinine ratio performed best, with areas under the ROC curves of 0.788, 0.795, and 0.814, respectively. By linear regression analysis, whole-urine TGFß, L1CAM, and urinary protein-to-creatinine ratio were associated with low GFR in the case-patients. CONCLUSIONS: Candidate biomarkers of obstructive nephropathy can be readily measured in whole urine and in urine exosomes. In boys with PUV, these biomarkers correlate with GFR.
Assuntos
Nefropatias/urina , Rim/fisiopatologia , Molécula L1 de Adesão de Célula Nervosa/urina , Fator de Crescimento Transformador beta/urina , Obstrução Uretral/complicações , Adolescente , Antígenos CD/urina , Aquaporina 2/urina , Área Sob a Curva , Biomarcadores/urina , Caderinas/urina , Estudos de Casos e Controles , Criança , Pré-Escolar , Creatinina/urina , Progressão da Doença , Exossomos/metabolismo , Estudos de Viabilidade , Taxa de Filtração Glomerular , Humanos , Lactente , Nefropatias/diagnóstico , Nefropatias/etiologia , Nefropatias/fisiopatologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/urina , Modelos Lineares , Modelos Logísticos , Masculino , Valor Preditivo dos Testes , Proteinúria/etiologia , Proteinúria/urina , Proteômica/métodos , Curva ROC , Canais de Cátion TRPV/urina , Obstrução Uretral/fisiopatologia , Obstrução Uretral/urina , Urinálise , ATPases Vacuolares Próton-Translocadoras/urina , beta Catenina/urinaRESUMO
Congenital urinary tract obstruction (diagnosed antenatally by ultrasound screening) is one of the main causes of end-stage kidney disease in children. The extent of kidney injury in early gestation and the resultant abnormality in kidney development determine fetal outcome and postnatal renal function. Unfortunately, the current approach to diagnostic evaluation of the severity of injury has inherently poor diagnostic and prognostic value because it is based on the assessment of fetal tubular function from fetal urine samples rather than on estimates of the dysplastic changes in the injured developing kidney. To improve the outcome in children with congenital urinary tract obstruction, new biomarkers reflecting these structural changes are needed. Genomic and proteomic techniques that have emerged in the past decade can help identify the key genes and proteins from biological fluids, including amniotic fluid, that might reflect the extent of injury to the developing kidney.
Assuntos
Nefropatias/diagnóstico , Nefropatias/etiologia , Rim/metabolismo , Doenças Urológicas/congênito , Doenças Urológicas/diagnóstico , Animais , Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Genômica , Idade Gestacional , Taxa de Filtração Glomerular , Humanos , Rim/embriologia , Rim/fisiopatologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/etiologia , Falência Renal Crônica/genética , Falência Renal Crônica/metabolismo , Valor Preditivo dos Testes , Prognóstico , Índice de Gravidade de Doença , Ultrassonografia Pré-Natal , Doenças Urológicas/complicações , Doenças Urológicas/metabolismo , Doenças Urológicas/terapiaRESUMO
The use of internal standards in the determination of volatile organic compounds as described in SW-846 Method 8260C introduces a potential for bias in results once the internal standards (ISTDs) are added to a sample for analysis. The bias is relative to the dissimilarity between the analyte and internal standard physical properties that influence how easily analytes are separated from a matrix and concentrated during analysis. Method 5032 is a vacuum distillation procedure for extracting analytes from a sample for use with Method 8260C. Vacuum distillation is also incorporated within another GC/MS analytical procedure, Method 8261A. Method 8260C/5032 and Method 8261A are experimentally identical, however, Method 8261A uses internal standards differently by relating the recovery of each compound to its boiling point and relative volatility. By processing each analysis (water, soil, and biota) using both Method 8260C and Method 8261A, the two approaches are compared on the basis of analyte bias and the failure rate of the quality controls. Analytes were grouped by how similar their boiling points and natural log of their relative volatilities (lnRVs) were to their Method 8260C recommended ISTDs. For the most similar analytes, the Method 8260C determinations yielded an average bias less than 10% and a failure to meet calibration criteria less than 7%. However, as the difference between analyte and ISTD became greater the bias increased to over 40% (matrix dependent) and its calibration failure rate approached 70%. In comparison, when the Method 8260C data were reprocessed as Method 8261A determinations, this trend for groupings was minimized with biases increasing from 6% to only 20% and the calibration failure rate went from 0% to 15%.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/normas , Compostos Orgânicos Voláteis/análise , Calibragem , Destilação , Padrões de Referência , Solo/química , Compostos Orgânicos Voláteis/química , Água/químicaRESUMO
The heat-stable antigen, CD24, is a cell-surface sialoglycoprotein expressed on immature cells that disappears after they have reached their final stage of differentiation. In mice, CD24 expression is preferentially upregulated in the developing mouse metanephros as compared with the surrounding intermediate mesoderm, but its role and expression in the developing human kidney has not been well described. Here we found in normal human fetal kidneys (8 to 38 weeks of gestation) that CD24 expression was upregulated and restricted to the early epithelial aggregates of the metanephric blastema and to the committed proliferating tubular epithelia of the S-shape bodies. Individual cells expressing CD24 were identified in the interstitium of later gestation and postnatal kidneys. In freshly isolated cells, FACS analysis identified distinct CD24(+) and CD24(+)133(+) cell populations constituting up to 16 and 14 percent, respectively, of the total cells analyzed. Early fetal urinary tract obstruction resulted in upregulation of CD24 expression both in developing epithelial structures of early stages and in the cells of the injured tubular epithelium of the later gestation kidneys. Our results highlight the cell-specific expression of CD24 in the developing human kidney and its dysregulation during fetal urinary tract obstruction.
Assuntos
Antígeno CD24/análise , Rim/crescimento & desenvolvimento , Regulação para Cima , Antígeno CD24/genética , Feto , Citometria de Fluxo , Idade Gestacional , Humanos , Rim/citologia , Rim/embriologia , Distribuição Tecidual , Obstrução UreteralRESUMO
Congenital urinary tract obstruction induces changes to the renal collecting duct epithelium, including alteration and depletion of intercalated cells. To study the effects of obstruction on the ontogeny of intercalated cell development, we examined normal and obstructed human fetal and postnatal kidneys. In the normal human fetal kidney, intercalated cells originated in the medullary collecting duct at 8 weeks gestation and remained most abundant in the inner medulla throughout gestation. In the cortex, intercalated cells were rare at 18 and 26 weeks gestation and observed at low abundance at 36 weeks gestation. Although early intercalated cells exhibit an immature phenotype, Type A intercalated cells predominated in the inner and outer medullae at 26 and 36 weeks gestation with other intercalated cell subtypes observed rarely. Postnatally, the collecting duct epithelium underwent a remodeling whereby intercalated cells become abundant in the cortex yet absent from the inner medulla. In 18-week obstructed kidneys with mild to moderate injury, the intercalated cells became more abundant and differentiated than the equivalent age-matched normal kidney. In contrast, more severely injured ducts of the late obstructed kidney exhibited a significant reduction in intercalated cells. These studies characterize the normal ontogeny of human intercalated cell development and suggest that obstruction induces premature remodeling and differentiation of the fetal collecting duct epithelium.
Assuntos
Epitélio/fisiologia , Feto/fisiologia , Túbulos Renais Coletores/embriologia , Túbulos Renais Coletores/fisiologia , Estudos de Casos e Controles , Contagem de Células , Diferenciação Celular/fisiologia , Proliferação de Células , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Epitélio/patologia , Feto/patologia , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Rim/citologia , Rim/embriologia , Rim/crescimento & desenvolvimento , Rim/patologia , Túbulos Renais Coletores/crescimento & desenvolvimento , Túbulos Renais Coletores/patologia , Modelos Biológicos , Regeneração/fisiologia , Obstrução Ureteral/congênito , Obstrução Ureteral/patologia , Doenças Urológicas/congênito , Doenças Urológicas/patologiaRESUMO
Epithelial-mesenchymal transition (EMT) has emerged in recent years as an important process in the development of organ fibrosis in many human diseases. Our previous experience in a nonhuman primate model of obstructive nephropathy suggested that EMT of collecting duct epithelium contributes to the development of interstitial fibrosis. In this study we demonstrate for the first time in humans that obstructed fetal collecting duct epithelium undergoes transition to mesenchymal phenotype, characterized by decreased expression of epithelial markers, de novo expression of mesenchymal markers with subsequent loss of cell-cell interaction, disruption of the basement membrane, and increased deposition of extracellular matrix into the expanded interstitium of the obstructed kidney. The results of this study therefore support the previous findings from animal studies and suggest that EMT of the collecting duct epithelium might contribute to the development of interstitial fibrosis in human fetal obstructive nephropathy.
Assuntos
Epitélio/imunologia , Epitélio/patologia , Túbulos Renais Coletores/imunologia , Túbulos Renais Coletores/patologia , Obstrução do Colo da Bexiga Urinária/congênito , Obstrução do Colo da Bexiga Urinária/patologia , Diferenciação Celular , Humanos , Fenótipo , Obstrução do Colo da Bexiga Urinária/imunologiaRESUMO
Benzene, a carcinogen that can cause cancer in humans, may form at nanogram per gram levels in some beverages containing both benzoate salts and ascorbic or erythorbic acids. Through a series of reactions, a hydroxyl radical forms that can decarboxylate benzoate to form benzene. Elevated temperatures and light stimulate these reactions, while sugar and ethylenediaminetetraacetic acid (EDTA) can inhibit them. A headspace gas chromatography/mass spectrometry method for the determination of benzene in beverages was developed and validated. The method was used to conduct a survey of 199 soft drinks and other beverages. The vast majority of beverages sampled contained either no detectable benzene or levels below the U.S. Environmental Protection Agency's drinking water limit of 5 ng/g. Beverages found to contain 5 ng/g benzene or more were reformulated by the manufacturers. The amount of benzene found in the reformulated beverages ranged from none detected to 1.1 ng/g.