RESUMO
Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Rim/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Regulação para Baixo , Rim/citologia , Rim/embriologia , Glomérulos Renais/citologia , Glomérulos Renais/embriologia , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/metabolismo , Transplante de Rim , Túbulos Renais/citologia , Túbulos Renais/embriologia , Túbulos Renais/crescimento & desenvolvimento , Túbulos Renais/metabolismo , Camundongos , Podócitos/citologia , Podócitos/metabolismo , RNA-Seq , Análise de Célula Única , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Multiciliated cells (MCCs) differentiate hundreds of motile cilia that beat to drive fluid movement over various kinds of epithelia. In Xenopus, mice and human, the coiled-coil containing protein Mcidas (Mci) has been shown to be a key transcriptional regulator of MCC differentiation. We have examined Mci function in the zebrafish, another model organism that is widely used to study ciliary biology. We show that zebrafish mci is expressed specifically in the developing MCCs of the kidney tubules, but surprisingly, not in those of the nasal placodes. Mci proteins lack a DNA binding domain and associate with the cell-cycle transcription factors E2f4/5 for regulating MCC-specific gene expression. We found that while the zebrafish Mci protein can complex with the E2f family members, its sequence as well as the requirement and sufficiency for MCC differentiation has diverged significantly from Mci homologues of the tetrapods. We also provide evidence that compared to Gmnc, another related coiled-coil protein that has recently been shown to regulate MCC development upstream of Mci, the Mci protein originated later within the vertebrate lineage. Based on these data, we argue that in contrast to Gmnc, which has a vital role in the genetic circuitry that drives MCC formation, the requirement of Mci, at least in the zebrafish, is not obligatory.
Assuntos
Cílios , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais/embriologia , Transdução de Sinais , Fatores de Transcrição , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Ciclo Celular , Cílios/genética , Cílios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.
Assuntos
Biologia Computacional , Infecções por Coronavirus/imunologia , Neoplasias/imunologia , Pneumonia Viral/imunologia , Adulto , Enzima de Conversão de Angiotensina 2 , Betacoronavirus , COVID-19 , Infecções por Coronavirus/complicações , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genótipo , Células Caliciformes/metabolismo , Hepatócitos/metabolismo , Humanos , Sistema Imunitário , Túbulos Renais/embriologia , Masculino , Neoplasias/complicações , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Prognóstico , RNA-Seq , SARS-CoV-2 , Transdução de SinaisRESUMO
Several life-threatening diseases of the kidney have their origins in mutational events that occur during embryonic development. In this study, we investigate the role of the Wolffian duct (WD), the earliest embryonic epithelial progenitor of renal tubules, in the etiology of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with a germline mutation of one of the two Pkd1 alleles. For the disease to occur, a second event that disrupts the expression of the other inherited Pkd1 allele must occur. We postulated that this secondary event can occur in the pronephric WD. Using Cre-Lox recombination, mice with WD-specific deletion of one or both Pkd1 alleles were generated. Homozygous Pkd1-targeted deletion in WD-derived tissues resulted in mice with large cystic kidneys and serologic evidence of renal failure. In contrast, heterozygous deletion of Pkd1 in the WD led to kidneys that were phenotypically indistinguishable from control in the early postnatal period. High-throughput sequencing, however, revealed underlying gene and microRNA (miRNA) changes in these heterozygous mutant kidneys that suggest a strong predisposition toward developing ADPKD. Bioinformatic analysis of this data demonstrated an upregulation of several miRNAs that have been previously associated with PKD; pathway analysis further demonstrated that the differentially expressed genes in the heterozygous mutant kidneys were overrepresented in signaling pathways associated with maintenance and function of the renal tubular epithelium. These results suggest that the WD may be an early epithelial target for the genetic or molecular signals that can lead to cyst formation in ADPKD.
Assuntos
Túbulos Renais/embriologia , Rim Policístico Autossômico Dominante/genética , Insuficiência Renal/genética , Canais de Cátion TRPP/genética , Ductos Mesonéfricos/patologia , Alelos , Animais , Modelos Animais de Doenças , Epitélio/embriologia , Epitélio/patologia , Feminino , Mutação em Linhagem Germinativa , Humanos , Túbulos Renais/patologia , Camundongos , Camundongos Knockout , Rim Policístico Autossômico Dominante/sangue , Rim Policístico Autossômico Dominante/complicações , Rim Policístico Autossômico Dominante/patologia , Insuficiência Renal/sangue , Insuficiência Renal/diagnóstico , Insuficiência Renal/patologia , Transdução de Sinais/genética , Ductos Mesonéfricos/embriologiaRESUMO
Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.
Assuntos
Túbulos Renais/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais/citologia , Túbulos Renais/embriologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas de Xenopus/análise , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl-/- mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl-/- mice died soon after birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl-/- neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl-/- neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.
Assuntos
Hidronefrose/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Glomérulos Renais/embriologia , Túbulos Renais/embriologia , Proteínas de Membrana/genética , Animais , Linhagem Celular , Feminino , Hidronefrose/mortalidade , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Glomérulos Renais/patologia , Túbulos Renais/patologia , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
INTRODUCTION: In transplantation medicine calcineurin inhibitors (CNI) still represent the backbone of immunosuppressive therapy. The nephrotoxic potential of the CNI Cyclosporine A (CsA) and Tacrolimus (FK506) is well recognized and CNI not only have been linked with toxicity, but also with cellular senescence which hinders parenchymal tissue regeneration and thus may prime kidneys for subsequent insults. To minimize pathological effects on kidney grafts, alternative immunosuppressive agents like mTOR inhibitors or the T-cell co-stimulation blocker Belatacept have been introduced. METHODS: We compared the effects of CsA, FK506 and Sirolimus on the process of cellular senescence in different human renal tubule cell types (HK2, RPTEC). Telomere length (by real time PCR), DNA synthesis (by BrdU incorporation), cell viability (by Resazurin conversion), gene expression (by RT-PCR), protein (by western blotting), Immuncytochemistry and H2O2 production (by Amplex Red® conversion) were evaluated. RESULTS: DNA synthesis was significantly reduced when cells were treated with cyclosporine but not with tacrolimus and sirolimus. Resazurin conversion was not altered by all three immunosuppressive agents. The gene expression as well as protein production of the cell cycle inhibitor p21 (CDKN1A) but not p16 (CDKN2A) was significantly induced by cyclosporine compared to the other two immunosuppressive agents when determined by western blotting an immuncytochemistry. Relative telomere length was reduced and hydrogen peroxide production increased after treatment with CsA but not with FK506 or sirolimus. CONCLUSION: In summary, renal tubule cells exposed to CsA show clear signs of cellular senescence where on the contrary the second calcineurin inhibitor FK506 and the mTOR inhibitor sirolimus are not involved in such mechanisms. Chronic renal allograft dysfunction could be in part triggered by cellular senescence induced by immunosuppressive medication and the choice of drug could therefore influence long term outcome. Tacrolimus and Sirolimus are equally effective in avoiding cellular senescence compared to cyclosporine at least in parts due to a lack of induction of reactive oxygen species.
Assuntos
Senescência Celular/efeitos dos fármacos , Ciclosporina/toxicidade , Células Epiteliais/efeitos dos fármacos , Imunossupressores/toxicidade , Rim/citologia , Sirolimo/toxicidade , Tacrolimo/toxicidade , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Transplante de Rim , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/embriologia , Espécies Reativas de Oxigênio/metabolismo , Encurtamento do Telômero/efeitos dos fármacosRESUMO
In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.
Assuntos
Divisão Celular , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Células Cultivadas , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Doenças Renais Císticas/patologia , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Morfogênese , Néfrons/metabolismo , Néfrons/patologia , Fuso Acromático/metabolismoRESUMO
The nephron is the functional subunit of the vertebrate kidney and plays important osmoregulatory and excretory roles during embryonic development and in adulthood. Despite its central role in kidney function, surprisingly little is known about the molecular and cellular processes that control nephrogenesis. The zebrafish pronephric kidney, comprising two nephrons, provides a visually accessible and genetically tractable model system for a better understanding of nephron formation. Using this system, various developmental processes, including the commitment of mesoderm to a kidney fate, renal tubule proliferation, and migration, can be studied during nephrogenesis. Here, we discuss some of these processes in zebrafish with a focus on the pathways that influence renal tubule cell morphogenesis.
Assuntos
Túbulos Renais/embriologia , Morfogênese/fisiologia , Organogênese/fisiologia , Peixe-Zebra/embriologia , AnimaisRESUMO
BACKGROUNDS/AIMS: Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown. METHODS: We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization. RESULTS: We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos. CONCLUSION: Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.
Assuntos
Túbulos Renais/crescimento & desenvolvimento , L-Gulonolactona Oxidase/análise , Pronefro/crescimento & desenvolvimento , Animais , Biomarcadores/análise , Feminino , Túbulos Renais/embriologia , Túbulos Renais/enzimologia , Mamíferos , Pronefro/embriologia , Pronefro/enzimologia , Alinhamento de Sequência , Xenopus laevisRESUMO
BACKGROUND: Wnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system. RESULTS: The Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11 (-/-) mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11 (-/-) mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency. CONCLUSIONS: The Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11 (-/-) mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.
Assuntos
Glomérulos Renais/anormalidades , Túbulos Renais/anormalidades , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Glomérulos Renais/embriologia , Túbulos Renais/embriologia , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Patients with von Hippel-Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. 'Clear cell' tumors contain large, proliferating cells with 'clear cytoplasm', and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl(-/-)) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl(-/-) zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl(-/-) zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl(-/-) pronephros is reminiscent of clear cell histology, indicating that the vhl(-/-) mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl(-/-) zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Pronefro/metabolismo , Pronefro/patologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Proliferação de Células , Vesículas Citoplasmáticas/metabolismo , Desenvolvimento Embrionário , Glicogênio/metabolismo , Humanos , Neoplasias Renais/metabolismo , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/ultraestrutura , Larva/metabolismo , Estadiamento de Neoplasias , Fenótipo , Pronefro/embriologia , Pronefro/ultraestruturaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disease that still lacks effective therapy. Repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins (BMPs) and a ligand for neogenin, is expressed in renal tubular epithelial cells. Previous studies showed that RGMb plays negative roles in several types of tumors and prevents the immune system from over activation. The present study was designed to explore the effects of RGMb in ADPKD development. We found that expression of RGMb in kidney was less in PKD mice than wild-type mice. With stimulation of 8-bromo-cAMP, RGMb-null embryonic kidneys had greater cyst index, though their ureteric bud branched less than wild-type mice at E13.5. Postnatal RGMb-null kidneys showed interstitial hyperplasia and decreased tubular structures, especially in the boundary area of renal cortex and medulla. RGMb overexpression dramatically inhibited cyst development and promoted tubulogenesis in MDCK cells grown in 3D collagen gels. Biochemical analysis showed increased p-Smad1/5/8 and decreased p-ERK in RGMb-overexpressing MDCK cells, suggesting modulated BMP signaling. Specific inhibition of p-Smad1/5/8 by LDN193189 reversed the suppression of RGMb on MDCK cyst model. These results reveal RGMb as a novel regulator for ADPKD by promoting renal tubule branching and regulating BMP signaling pathway. Elevating RGMb and enhancing p-Smad1/5/8 are promising new strategies to treat ADPKD.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais , Proliferação de Células/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila melanogaster/metabolismo , Túbulos Renais/metabolismo , Microvilosidades/metabolismo , Animais , Transporte Biológico , Moléculas de Adesão Celular Neuronais/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais/embriologia , Microtúbulos/metabolismoRESUMO
UNLABELLED: Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1. Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease. KEY MESSAGE: Renal cyst growth depends on glucose concentration in two in vitro cyst models. High glucose leads to upregulation of the calcium-activated chloride channel ANO1. High glucose promotes calcium-activated chloride secretion via ANO1. Glucose-dependent secretion can be inhibited by a specific inhibitor of ANO1.
Assuntos
Cloretos/metabolismo , Cistos/patologia , Glucose/farmacologia , Túbulos Renais/patologia , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/genética , Animais , Anoctamina-1 , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , AMP Cíclico/metabolismo , Cães , Túbulos Renais/citologia , Túbulos Renais/embriologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Transcricional/genéticaRESUMO
Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Túbulos Renais/embriologia , Organogênese/genética , Pronefro/embriologia , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Anormalidades Múltiplas/genética , Animais , Animais Geneticamente Modificados , Padronização Corporal/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/biossíntese , Meningocele/genética , Mesoderma/embriologia , Modelos Animais , Morfolinos/genética , Organogênese/fisiologia , Região Sacrococcígea/anormalidades , Fatores de Transcrição/biossíntese , Proteínas de Peixe-Zebra/biossínteseRESUMO
The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and maintenance, its exact molecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either lineage results in abnormal tubulogenesis, with profound defects in polarity, lumen formation and the actin cytoskeleton. Ultimately, these defects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processes by regulating the polarity Par complex (Par3-Par6-aPKC-Cdc42) and the cytoskeletal proteins N-Wasp and ezrin. Thus, we conclude that the principal role of Cdc42 in ureteric bud and metanephric mesenchyme development is to regulate epithelial cell polarity and the actin cytoskeleton.
Assuntos
Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais/embriologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Citoesqueleto/genética , Células Epiteliais/citologia , Camundongos , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Túbulos Renais/embriologia , Pronefro/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Padronização Corporal/fisiologia , Contagem de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Pronefro/citologia , Pronefro/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Tretinoína/metabolismo , Tretinoína/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
When a tubular structure forms during early embryogenesis, tubular elongation and lumen formation (epithelialization) proceed simultaneously in a spatiotemporally coordinated manner. We here demonstrate, using the Wolffian duct (WD) of early chicken embryos, that this coordination is regulated by the expression of FGF8, which shifts posteriorly during body axis elongation. FGF8 acts as a chemoattractant on the leader cells of the elongating WD and prevents them from epithelialization, whereas static ('rear') cells that receive progressively less FGF8 undergo epithelialization to form a lumen. Thus, FGF8 acts as a binary switch that distinguishes tubular elongation from lumen formation. The posteriorly shifting FGF8 is also known to regulate somite segmentation, suggesting that multiple types of tissue morphogenesis are coordinately regulated by macroscopic changes in body growth.
Assuntos
Epitélio/embriologia , Epitélio/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Túbulos Renais/citologia , Túbulos Renais/embriologia , Organogênese , Animais , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Embrião de Galinha , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/embriologia , Mesoderma/metabolismo , Modelos Biológicos , Organogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ductos Mesonéfricos/citologia , Ductos Mesonéfricos/efeitos dos fármacos , Ductos Mesonéfricos/embriologia , Ductos Mesonéfricos/metabolismo , Proteínas ras/metabolismoRESUMO
Recent advances in renal tissue engineering have shown that dissociated, early renogenic tissue from the developing embryo can self-assemble into morphologically accurate kidney-like organs arranged around a central collecting duct tree. In order for such self-assembled kidneys to be useful therapeutically or as models for drug screening, it is necessary to demonstrate that they are functional. One of the main functional characteristics of mature kidneys is transport of organic anions and cations into and out of the proximal tubule. Here, we show that the transport function of embryonic kidneys allowed to develop in culture follows a developmental time-course that is comparable to embryonic kidney development in vivo. We also demonstrate that serially-reaggregated engineered kidneys can transport organic anions and cations through specific uptake and efflux channels. These results support the physiological relevance of kidneys grown in culture, a commonly used model for kidney development and research, and suggest that serially-reaggregated kidneys self-assembled from separated cells have some functional characteristics of intact kidneys.