RESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the presence of numerous fluid-filled cysts, extensive fibrosis, and the progressive decline in kidney function. Transforming growth factor-ß1 (TGF-ß1), an important mediator for renal fibrosis and chronic kidney disease, is overexpressed by cystic cells compared with normal kidney cells; however, its role in PKD pathogenesis remains undefined. To investigate the effect of TGF-ß1 on cyst growth, fibrosis, and disease progression, we overexpressed active TGF-ß1 specifically in collecting ducts (CDs) of phenotypic normal (Pkd1RC/+) and Pkd1RC/RC mice. In normal mice, CD-specific TGF-ß1 overexpression caused tubule dilations by 5 wk of age that were accompanied by increased levels of phosphorylated SMAD3, α-smooth muscle actin, vimentin, and periostin; however, it did not induce overt cyst formation by 20 wk. In Pkd1RC/RC mice, CD overexpression of TGF-ß1 increased cyst epithelial cell proliferation. However, extensive fibrosis limited cyst enlargement and caused contraction of the kidneys, leading to a loss of renal function and a shortened lifespan of the mice. These data demonstrate that TGF-ß1-induced fibrosis constrains cyst growth and kidney enlargement and accelerates the decline of renal function, supporting the hypothesis that a combined therapy that inhibits renal cyst growth and fibrosis will be required to effectively treat ADPKD.
Assuntos
Rim/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Fibrose , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/fisiopatologia , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Fatores de Tempo , Fator de Crescimento Transformador beta1/genéticaRESUMO
The goal of this study was to determine whether transforming growth factor-ß1 (TGF-ß1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-ß1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-ß1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-ß1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-ß1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-ß1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-ß1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-ß1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-ß1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-ß1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-ß1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-ß1, which further suggests that TGF-ß1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-ß1, via TGF-ß1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Infertilidade Masculina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ducto Deferente/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Ânions/metabolismo , Anisomicina/farmacologia , Benzamidas/farmacologia , Células Cultivadas , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Dioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Inibidores da Síntese de Proteínas/farmacologia , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R(TE), a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R(TE) significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Ducto Deferente/citologia , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Claudinas/metabolismo , Fenômenos Eletrofisiológicos , Células Epiteliais/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Proteína Quinase 11 Ativada por Mitógeno/antagonistas & inibidores , Modelos Animais , Ocludina , Técnicas de Patch-Clamp , Suínos , Junções Íntimas/metabolismoRESUMO
Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR(-/-) and CFTR(ΔF508/ΔF508) neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR(-/-) monolayers can exhibit Na(+) reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl(-) and/or HCO(3)(-) transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/patologia , Modelos Animais de Doenças , Epididimo/anormalidades , Suínos , Ducto Deferente/anormalidades , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Ânions/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Masculino , Muco/metabolismo , Fenótipo , Ducto Deferente/metabolismoRESUMO
Testosterone induces and maintains prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2) expression in vas deferens epithelial cells, but it remains unknown whether this has a physiological role in the context of male reproductive biology. Prostaglandins induce concentration-dependent increases in anion secretion in porcine vas deferens epithelial cell (1 degrees PVD) monolayers, where bicarbonate contributes to cAMP-stimulated anion secretion. Moreover, bradykinin induces anion secretion across 1 degrees PVD monolayers that is indomethacin sensitive, and both PTGS2 and PTGS1 are expressed in this model system. Therefore, it was hypothesized that testosterone modulates anion secretion across vas deferens epithelia via PTGS-dependent pathways and prostaglandin synthesis. Porcine vas deferens epithelial cells were isolated and cultured as monolayers on permeable supports until assayed in modified Ussing chambers. RNA and protein were isolated concurrently for semiquantitative expression analysis. Testosterone upregulated basal and bradykinin-induced short-circuit current across 1 degrees PVD monolayers, indicative of anion secretion. Testosterone also induced greater transepithelial electrical resistance. Increases in anion secretion were associated with preferential upregulation of PTGS2 at the mRNA and protein levels. In addition, testosterone induced greater basal and bradykinin-induced anion secretion across vas deferens epithelial cells isolated from the distal segment of the duct. Taken together, these results suggest that testosterone upregulates epithelial responsiveness to acute modulations of anion secretion (likely bicarbonate secretion), which ultimately modifies the environment to which sperm are exposed.
Assuntos
Ânions/metabolismo , Transporte de Íons , Prostaglandina-Endoperóxido Sintases/metabolismo , Testosterona/metabolismo , Ducto Deferente/metabolismo , Animais , Bradicinina/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/metabolismo , Masculino , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Suínos , Regulação para CimaRESUMO
Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.
Assuntos
Ânions/metabolismo , Linhagem Celular/fisiologia , Epitélio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Ducto Deferente/citologia , Animais , Colforsina/farmacologia , Epitélio/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Neurotransmissores/metabolismo , Suínos , Ducto Deferente/metabolismoRESUMO
During vertebrate development the gonad has two possible fates, the testis or the ovary. The choice between these fates is made by a variety of sex-determining mechanisms, from the sex-determining gene on the Y chromosome (Sry) in mammals, to nongenetic temperature-dependent systems in many reptiles. Despite the differences in the mechanisms at the top of the sex-determining cascade, the resulting morphology and many genes involved in early testis and ovarian development are common to most vertebrates, leading to the hypothesis that the underlying processes of sex determination are conserved. In this study, we examined the early steps of gonad development in the red-eared slider turtle (Trachemys scripta), a species that uses the temperature of egg incubation to determine sex. A dramatic increase in cell proliferation was observed in the male gonad during the earliest stages of sex determination. Using the localization of Wilms' Tumor suppressor 1 (WT1), we determined that this proliferation increase occurred in a population that contained pre-Sertoli cells. The proliferation of pre-Sertoli cells has been documented during sex determination in both mice and alligators, suggesting that proliferation of this cell type has an important role in vertebrate testis organogenesis and the determination of male fate.