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OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney condition, accounting for 7%-10% of patients with kidney failure. Fundamental basic science and clinical research on ADPKD is underway worldwide but no one has yet considered which areas should be prioritised to maximise returns from limited future funding. The Polycystic Kidney Disease Charity began a priority setting partnership with the James Lind Alliance (JLA) in the UK in 2019-2020 to identify areas of uncertainty in the ADPKD care pathway and allow patients, carers and healthcare professionals to rank the 10 most important questions for research. DESIGN: The scope covered ADPKD diagnosis and management, identifying new treatments to prevent/slow disease progression and practical, integrated patient support (https://pkdcharity.org.uk/research/for-researchers/adpkd-research-priorities). We used adapted JLA methodology. Initially, an independent information specialist collated uncertainties in ADPKD care from recent consensus conference proceedings and additional literature. These were refined into indicative questions with Steering Group oversight. Finally, the 10 most important questions were established via a survey and online consensus workshop. SETTING: UK. PARTICIPANTS: 747 survey respondents (76% patients, 13% carers, 11% healthcare professionals); 23 workshop attendees. RESULTS: 117 uncertainties in ADPKD care were identified and refined into 35 indicative questions. A shortlist of 17 questions was established through the survey. Workshop participants reached agreement on the top 10 ranking. The top three questions prioritised by patients, carers and healthcare professionals centred around slowing disease progression, identifying persons for early treatment and organising care to improve outcomes. CONCLUSIONS: Our shortlist reflects the varied physical, psychological and practical challenges of living with and treating ADPKD, and perceived gaps in knowledge that impair optimal care. We propose that future ADPKD research funding takes these priorities into account to focus on the most important areas and to maximise improvements in ADPKD outcomes.
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Pesquisa Biomédica , Rim Policístico Autossômico Dominante , Cuidadores , Progressão da Doença , Prioridades em Saúde , Humanos , Rim Policístico Autossômico Dominante/terapia , Reino UnidoRESUMO
ADPKD is the most common genetic disease of the kidney leading to end-stage renal disease necessitating renal replacement therapy at any time between the 1st and 8th decades of life due to widely variable rates of disease progression. This presents significant patient anxiety and a significant prognostic and therapeutic challenge. Tolvaptan is the only approved drug licensed to slow ADPKD progression by reducing renal cystic expansion but side-effects can limit its efficacy. To address the need to identify new biomarkers to monitor progression of ADPKD and to evaluate the therapeutic effects of Tolvaptan, proteomic analysis was conducted on defined (40-100nm) urinary exosomes isolated from ADPKD patients phenotyped and clinically monitored over a 10-year period. Comparative Gene Ontology analysis of Tandem Mass Tag labelled mass spectrometry-derived protein profiles from urinary exosomes from ADPKD patients with rapid (>10ml/min/5 years decline in estimated glomerular filtration rate) versus slow progression showed distinctive patterns of pathway up-regulation. Clear discrimination between rapid and slowly-progressive profiles were seen in all stages functional decline in ADPKD patients whether with mild (>70ml/min), moderate (50-69ml/min) or severe (<49ml/min) disease at onset. Discriminatory pathways and proteins included Notch-, integrin- and growth factor-signalling; microtubular kinase, vesicular proteins and epidermal growth factor substrates. Confocal microscopy of fluorescently-labelled normal versus ADPKD epithelial cell-derived exosomes in vitro also identified ADPKD-dependent abnormalities in intracellular vesicular trafficking and implicated changes in ADPKD-dependent exosome secretion and target cell uptake as factors underlying urinary exosome excretion biomarker properties. Comparative proteomic analysis of urinary exosomal proteins in individual patients before and after treatment with Tolvaptan for 4 years also identified distinct patterns of pathway modification dependent on the degree of effectiveness of the therapeutic response. Up-regulation of Wnt-pathway and vesicular proteins were characteristic of urinary exosomes from ADPKD patients with good responses to Tolvaptan while upregulation of angiogenesis pathways and additional molecular forms of vasopressin receptor AVPR2 were characteristic in urinary exosomes of ADPKD patients with poor responses. Taken together, these studies conclude that proteomic profiling of urinary exosome biomarkers provides a specific, sensitive and practical non-invasive method to identify and monitor the rate of disease progression and the effects of Tolvaptan therapy in individual ADPKD patients. This provides a means to identify those patients most likely to benefit maximally from therapy and to progress towards a personalization of ADPKD prognosis and management.
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Retinoic acid (RA) activates RA receptors (RAR), resulting in RA response element (RARE)-dependent gene expression in renal collecting duct (CD). Emerging evidence supports a protective role for this activity in acute kidney injury (AKI) and chronic kidney disease (CKD). Herein, we examined this activity in RARE-LacZ transgenic mice and by RARE-Luciferase reporter assays in CD cells, and investigated how this activity responds to neurotransmitters and mediators of kidney injury. In RARE-LacZ mice, Adriamycin-induced heavy albuminuria was associated with reduced RA/RAR activity in CD cells. In cultured CD cells, RA/RAR activity was repressed by acetylcholine, albumin, aldosterone, angiotensin II, high glucose, cisplatin and lipopolysaccharide, but was induced by aristolochic acid I, calcitonin gene-related peptide, endothelin-1, gentamicin, norepinephrine and vasopressin. Compared with age-matched normal human CD cells, CD-derived renal cystic epithelial cells from patients with autosomal recessive polycystic kidney disease (ARPKD) had significantly lower RA/RAR activity. Synthetic RAR agonist RA-568 was more potent than RA in rescuing RA/RAR activity repressed by albumin, high glucose, angiotensin II, aldosterone, cisplatin and lipopolysaccharide. Hence, RA/RAR in CD cells is a convergence point of regulation by neurotransmitters and mediators of kidney injury, and may be a novel therapeutic target.
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Nefropatias/metabolismo , Túbulos Renais Coletores/metabolismo , Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Acetilcolina/farmacologia , Albuminas/farmacologia , Aldosterona/farmacologia , Angiotensina II/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Linhagem Celular , Cisplatino/farmacologia , Endotelina-1/farmacologia , Feminino , Glucose/farmacologia , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Vasopressinas/farmacologiaRESUMO
Biomarkers, the measurable indicators of biological conditions, are fast becoming a popular approach in providing information to track disease processes that could lead to novel therapeutic interventions for chronic conditions. Inherited, chronic kidney disease affects millions of people worldwide and although pharmacological treatments exist for some conditions, there are still patients whose only option is kidney dialysis and kidney transplantation. In the past 10 years, certain chronic kidney diseases have been reclassified as ciliopathies. Cilia in the kidney are antenna-like, sensory organelles that are required for signal transduction. One of the signalling pathways that requires the primary cilium in the kidney is Wnt signalling and it has three components such as canonical Wnt, non-canonical Wnt/planar cell olarity (PCP) and non-canonical Wnt/Ca2+ signalling. Identification of the novel role of ATM INteractor (ATMIN) as an effector molecule in the non-canonical Wnt/PCP pathway has intrigued us to investigate its potential role in chronic kidney disease. ATMIN could thus be an important biomarker in disease prognosis and treatment that might lighten the burden of chronic kidney disease and also affect on its progression.
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Biomarcadores/metabolismo , Cílios/metabolismo , Nefropatias/metabolismo , Doenças Renais Policísticas/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Progressão da Doença , Humanos , Nefropatias/terapia , Modelos Biológicos , Doenças Renais Policísticas/terapia , PrognósticoRESUMO
INTRODUCTION: Polycystic kidney disease (PKD) is a common genetic disease in which renal enlargement and loss of function is caused by progressive expansion of tubular cysts. To reverse the detrimental effects of PKD gene mutation(s) and to slow cystic expansion, new drug therapies are required. AREAS COVERED: The underlying cell biology leading to identification of molecular targets for PKD is reviewed. Specific focus is on studies published at the early pre-clinical level. These include genetic and epigenetic modulators, and drugs to slow cystic expansion and disease progression. Discussion of specific drugs and clinical trials is not within the scope of this article. Literature research methods included EndNote and PubMed online searches using keyword combinations: polycystic kidneys disease, pre-clinical, molecular targets, signal transduction, genetic modulators, epigenetic, therapeutic, receptors, kinases. Where possible, the most recent citations concerning a given target are referenced. EXPERT OPINION: It is suggested that the most promising targets for future therapeutic development are those that target upstream signaling events at cell membranes, such as the vasopressin-2 receptor (AVPR2), EGFR/ErbB2, and the ß-1-integrin receptor, as well as the intracellular integrator kinase, c-Src.
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Desenho de Fármacos , Terapia de Alvo Molecular , Doenças Renais Policísticas/tratamento farmacológico , Animais , Membrana Celular/metabolismo , Progressão da Doença , Epigênese Genética , Humanos , Mutação , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/fisiopatologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is clinically heterogenic. Biomarkers are needed to predict prognosis and guide management. We aimed to profile microRNA (miRNA) in ADPKD to gain molecular insight and evaluate biomarker potential. METHODS: Small-RNA libraries were generated from urine specimens of ADPKD patients (N = 20) and patients with chronic kidney disease of other etiologies (CKD, N = 20). In this report, we describe the miRNA profiles and baseline characteristics. For reference, we also examined the miRNA transcriptome in primary cultures of ADPKD cyst epithelia (N = 10), normal adult tubule (N = 8) and fetal tubule (N = 7) epithelia. RESULTS: In primary cultures of ADPKD kidney cells, miRNA cistrons mir-143(2) (9.2-fold), let-7i(1) (2.3-fold) and mir-3619(1) (12.1-fold) were significantly elevated compared to normal tubule epithelia, whereas mir-1(4) members (19.7-fold), mir-133b(2) (21.1-fold) and mir-205(1) (3.0-fold) were downregulated (P<0.01). Expression of the dysregulated miRNA in fetal tubule epithelia resembled ADPKD better than normal adult cells, except let-7i, which was lower in fetal cells. In patient biofluid specimens, mir-143(2) members were 2.9-fold higher in urine cells from ADPKD compared to other CKD patients, while expression levels of mir-133b(2) (4.9-fold) and mir-1(4) (4.4-fold) were lower in ADPKD. We also noted increased abundance mir-223(1) (5.6-fold), mir-199a(3) (1.4-fold) and mir-199b(1) (1.8-fold) (P<0.01) in ADPKD urine cells. In ADPKD urine microvesicles, miR-1(2) (7.2-fold) and miR-133a(2) (11.8-fold) were less abundant compared to other CKD patients (P<0.01). CONCLUSIONS: We found that in ADPKD urine specimens, miRNA previously implicated as kidney tumor suppressors (miR-1 and miR-133), as well as miRNA of presumed inflammatory and fibroblast cell origin (miR-223/miR-199), are dysregulated when compared to other CKD patients. Concordant with findings in the primary tubule epithelial cell model, this suggests roles for dysregulated miRNA in ADPKD pathogenesis and potential use as biomarkers. We intend to assess prognostic potential of miRNA in a followup analysis.
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Epigênese Genética , Túbulos Renais/metabolismo , MicroRNAs/genética , Rim Policístico Autossômico Dominante/genética , Urotélio/metabolismo , Adulto , Idoso , Biomarcadores/urina , Progressão da Doença , Feminino , Feto , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Túbulos Renais/patologia , Masculino , MicroRNAs/urina , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/urina , Cultura Primária de Células , Prognóstico , Urotélio/patologiaRESUMO
BACKGROUND: Vitamin A is necessary for kidney development and has also been linked to regulation of solute and water homeostasis and to protection against kidney stone disease, infection, inflammation, and scarring. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively. METHODOLOGY/PRINCIPAL FINDINGS: To explore target genes of endogenous tRA/RARs, we employed the mIMCD-3 mouse inner medullary CD cell line, which is a model of CD principal cells and exhibits constitutive tRA/RAR activity as CD principal cells do in vivo. Combining antagonism of RARs, inhibition of tRA synthesis, exposure to exogenous tRA, and gene expression profiling techniques, we have identified 125 genes as candidate targets and validated 20 genes that were highly regulated (Dhrs3, Sprr1a, and Ppbp were the top three). Endogenous tRA/RARs were more important in maintaining, rather than suppressing, constitutive gene expression. Although many identified genes were expressed in UBs and/or CDs, their exact functions in this cell lineage are still poorly defined. Nevertheless, gene ontology analysis suggests that these genes are involved in kidney development, renal functioning, and regulation of tRA signaling. CONCLUSIONS/SIGNIFICANCE: A rigorous approach to defining target genes for endogenous tRA/RARs has been established. At the pan-genomic level, genes regulated by endogenous tRA/RARs in a CD cell line have been catalogued for the first time. Such a catalogue will guide further studies on molecular mediators of endogenous tRA/RARs during kidney development and in relation to renal defects associated with vitamin A deficiency.
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Túbulos Renais Coletores/metabolismo , Receptores do Ácido Retinoico/metabolismo , Animais , Caderinas/metabolismo , Linhagem da Célula , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Imuno-Histoquímica/métodos , Inflamação , Rim/embriologia , Camundongos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Projetos Piloto , Elementos de Resposta , Transdução de Sinais , Tretinoína/metabolismo , Ureter/embriologia , Vitamina A/metabolismoAssuntos
Doenças Renais Policísticas/etiologia , Doenças Renais Policísticas/genética , Progressão da Doença , Humanos , Mutação , Doenças Renais Policísticas/metabolismo , Rim Policístico Autossômico Dominante/etiologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Epithelial cell polarity is essential for the establishment and maintenance of morphological and functional asymmetries that underlie normal renal structure and function and are brought about by the appropriate delivery of growth factor receptors and ion and fluid transporters and channels to apical or basolateral cell membranes. The fundamental process of cellular polarization is established early during development and is controlled by sets of evolutionarily conserved proteins that integrate intrinsic and extrinsic polarity cues. Specialized structural domains between adjacent cells and cells with their matrix, termed adherens junctions (AJ) and focal adhesions (FA), respectively, are formed that contain specific components of multi-molecular complexes acting as sites to recruit proteins and to activate intracellular mechano-transduction pathways. Regulation of these processes results in tight spatio-temporal control of renal tubule growth and lumen diameter. Abnormalities in macromolecular polarization complexes lead to a variety of diseases in different organs, a common example of which is Polycystic Kidney Disease (PKD), where epithelial cysts replace normal renal tubules. Membrane protein polarity defects in Autosomal Dominant (AD) PKD include the mis-polarization of normally basolateral membrane proteins to apical, lumenal membranes, such as epidermal growth factor (EGFR/ErbB) receptors and Na(+)K(+)-ATPase-α1 subunit; mis-polarization of normally apical membrane proteins to basolateral membranes, including the Na(+)K(+)2Cl(-) (NKCC1) symporter; and the failure to traffic and insert proteins into membranes resulting in their intracellular accumulation, such as E-cadherin and the ß1 subunit of Na(+)K(+)-ATPase. Abnormalities in structural AJ, FA and polarity complexes in ADPKD epithelia include loss of E-cadherin, and focal adhesion kinase (FAK), MALS-3, Crb and Dlg complexes as well as disruptions in Rab/sec and syntaxin trafficking and membrane docking pathways. Since proper polarization of epithelial cells lining renal tubules is essential for normal kidney development and differentiation to prevent abnormal cystic dilation, interventions to reverse polarity defects to normal would offer therapeutic opportunities for PKD. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
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Polaridade Celular/fisiologia , Rim/metabolismo , Rim/patologia , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Animais , Membrana Celular/metabolismo , Polaridade Celular/genética , Centríolos/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Espaço Intracelular/metabolismo , Rim/crescimento & desenvolvimento , Camundongos , Modelos Biológicos , Doenças Renais Policísticas/genética , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/metabolismo , Rim Policístico Autossômico Recessivo/patologia , Transdução de SinaisRESUMO
Angiogenesis is a fundamental step in several important physiological events and pathological conditions including embryonic development, wound repair, tumor growth and metastasis. PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX has also been shown to be phylogenetically and functionally distinct from PKA. This study presents the first evidence that PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation, which are the three essential processes for angiogenesis. In contrast, classic PKA demonstrated an inhibitory effect on endothelia vascular-like structure formation. Our findings suggest that PRKX is an important protein kinase engaged in the regulation of angiogenesis and could play critical roles in various physiological and pathological conditions involving angiogenesis. PRKX binds to Pin-1, Magi-1 and Bag-3, which regulate cell proliferation, apoptosis, differentiation and tumorigenesis. The interaction of PRKX with Pin-1, Magi-1 and Bag-3 could contribute to the stimulating role of PRKX in angiogenesis.
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Movimento Celular , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Adesão Celular , Células Cultivadas , Humanos , Camundongos , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)ß(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.
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Compostos de Anilina/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Nitrilas/farmacologia , Rim Policístico Autossômico Dominante/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Quinolinas/farmacologia , Animais , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Túbulos Renais Coletores/enzimologia , Túbulos Renais Coletores/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fosforilação , Rim Policístico Autossômico Dominante/enzimologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fatores de TempoRESUMO
Members of the epidermal growth factor (EGF) family bind to ErbB (EGFR) family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na(+) entry is mediated by the epithelial Na(+) channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
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Fator de Crescimento Epidérmico/fisiologia , Doenças Renais Policísticas/patologia , Doenças Renais Policísticas/fisiopatologia , Sódio/metabolismo , Animais , Proliferação de Células , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/fisiologia , Receptores ErbB/fisiologia , Humanos , Transporte de Íons , Rim/patologia , Rim/fisiopatologia , Modelos Biológicos , Doenças Renais Policísticas/etiologia , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/fisiopatologia , Rim Policístico Autossômico Recessivo/patologia , Rim Policístico Autossômico Recessivo/fisiopatologia , Canais de Cátion TRPP/fisiologiaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, and the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ. The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
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Proteínas Tirosina Fosfatases Semelhantes a Receptores/química , Canais de Cátion TRPP/química , Sequência de Aminoácidos , Animais , Caderinas/química , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/química , Humanos , Técnicas In Vitro , Rim/metabolismo , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Autosomal recessive polycystic kidney disease (ARPKD) is a significant hereditary renal disease occurring in infancy and childhood, which presents with greatly enlarged echogenic kidneys, ultimately leading to renal insufficiency and end-stage renal disease. ARPKD is caused by mutations in a single gene PKHD1, which encodes fibrocystin/polyductin (FPC), a large single transmembrane protein generally known to be on the primary cilium, basal body and plasma membrane. Here, using our newly generated antibody raised against the entire C-terminal intracellular cytoplasmic domain (ICD) of FPC, as well as our previously well-characterized antibody against a peptide of ICD, we report for the first time that at least one isoform of FPC is localized to the centrosome and mitotic spindle of dividing cells in multiple cell lines, including MDCK, mIMCD3, LLC-PK1, HEK293, RCTEC and HFCT cells. Using short-hairpin-mediated RNA interference, we show that the inhibition of FPC function in MDCK and mIMCD3 cells leads to centrosome amplification, chromosome lagging and multipolar spindle formation. Consistent with our in vitro findings, we also observed centrosome amplification in the kidneys from human ARPKD patients. These findings demonstrate a novel function of FPC in centrosome duplication and mitotic spindle assembly during cell division. We propose that mitotic defects due to FPC dysfunction contribute to cystogenesis in ARPKD.
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Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/metabolismo , Fuso Acromático/metabolismo , Adolescente , Animais , Linhagem Celular Tumoral , Centrossomo/metabolismo , Criança , Pré-Escolar , Modelos Animais de Doenças , Cães , Feminino , Humanos , Lactente , Masculino , Camundongos , Rim Policístico Autossômico Recessivo/genética , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Fuso Acromático/genéticaRESUMO
The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.
Assuntos
Proteínas de Transporte/fisiologia , Células Epiteliais/patologia , Doenças Renais Policísticas/patologia , Animais , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina , HumanosRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited renal disorder caused by defects in the PKD1 or PKD2 genes. ADPKD is associated with significant morbidity, and is a major underlying cause of end-stage renal failure (ESRF). Commonly, treatment options are limited to the management of hypertension, cardiovascular risk factors, dialysis, and transplantation when ESRF develops, although several new pharmacotherapies, including rapamycin, have shown early promise in animal and human studies. Evidence implicates polycystin-1 (PC-1), the gene product of the PKD1 gene, in regulation of the mTOR pathway. Here we demonstrate a mechanism by which the intracellular, carboxy-terminal tail of polycystin-1 (CP1) regulates mTOR signaling by altering the subcellular localization of the tuberous sclerosis complex 2 (TSC2) tumor suppressor, a gatekeeper for mTOR activity. Phosphorylation of TSC2 at S939 by AKT causes partitioning of TSC2 away from the membrane, its GAP target Rheb, and its activating partner TSC1 to the cytosol via 14-3-3 protein binding. We found that TSC2 and a C-terminal polycystin-1 peptide (CP1) directly interact and that a membrane-tethered CP1 protects TSC2 from AKT phosphorylation at S939, retaining TSC2 at the membrane to inhibit the mTOR pathway. CP1 decreased binding of 14-3-3 proteins to TSC2 and increased the interaction between TSC2 and its activating partner TSC1. Interestingly, while membrane tethering of CP1 was required to activate TSC2 and repress mTOR, the ability of CP1 to inhibit mTOR signaling did not require primary cilia and was independent of AMPK activation. These data identify a unique mechanism for modulation of TSC2 repression of mTOR signaling via membrane retention of this tumor suppressor, and identify PC-1 as a regulator of this downstream component of the PI3K signaling cascade.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/metabolismo , Adulto , Linhagem Celular , Humanos , Immunoblotting , Imuno-Histoquímica , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Serina/metabolismo , Serina-Treonina Quinases TOR , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genética , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na(+) absorption. Epidermal growth factor (EGF) is involved in the regulation of Na(+) transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na(+) absorption. The aim of the present study was to characterize the EGF regulation of Na(+) transport in cultured mouse renal collecting duct principal mpkCCD(c14) cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na(+) transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF-alpha, HB-EGF, and amphiregulin which also belong to the EGF-related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na(+) reabsorption. Inhibition of PI3-kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD(c14) cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague-Dawley rat kidneys. Both acute and long-term effects of EGF were abolished when cells were treated with tyrphostin AG-825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na(+) transport and that EGF biphasically modulates sodium transport in mpkCCD(c14) cells via the ErbB2 receptor.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Canais Epiteliais de Sódio/metabolismo , Glicoproteínas/metabolismo , Túbulos Renais Coletores/metabolismo , Receptor ErbB-2/metabolismo , Sódio/metabolismo , Amilorida/farmacologia , Anfirregulina , Animais , Benzotiazóis/farmacologia , Western Blotting , Butadienos/farmacologia , Linhagem Celular , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Família de Proteínas EGF , Canais Epiteliais de Sódio/efeitos dos fármacos , Flavonoides/farmacologia , Glicoproteínas/antagonistas & inibidores , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transporte de Íons , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Cinética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Camundongos , Morfolinas/farmacologia , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Fator de Crescimento Transformador alfa/metabolismo , Tirfostinas/farmacologiaRESUMO
Integrin-associated focal adhesion complex formation and turnover plays an essential role in directing interactions between epithelial cells and the extracellular matrix during organogenesis, leading to appropriate cell spreading, cell-matrix adhesion, and migration. Autosomal recessive polycystic kidney disease (ARPKD) is associated with loss of function of PKHD1-encoded protein fibrocystin-1 and is characterized by cystic dilation of renal collecting tubules (CT) in utero and loss of renal function in patients if they survive the perinatal period. Normal polycystin-1 (PC-1)/focal adhesion complex function is required for control of CT diameter during renal development, and abnormalities in these complexes have been demonstrated in human PC-1 mutant cystic cells. To determine whether loss of fibrocystin-1 was associated with focal adhesion abnormalities, ARPKD cells or normal age-matched human fetal (HF)CT cells in which fibrocystin-1 had been decreased by 85% by small interfering RNA inhibition were compared with normal HFCT. Accelerated attachment and spreading on collagen matrix and decreased motility of fibrocystin-1-deficient cells were associated with longer paxillin-containing focal adhesions, more complex actin-cytoskeletal rearrangements, and increased levels of total beta(1)-integrin, c-Src, and paxillin. Immunoblot analysis of adhesive cells using site-specific phospho-antibodies demonstrated ARPKD-associated loss of activation of focal adhesion kinase (FAK) by phosphorylation at its autophosphorylation site (Y397); accelerated FAK inhibition by phosphorylation at Y407, S843, and S910; as well as increased activation of c-Src at pY418. Paxillin coimmunoprecipitation analyses suggested that fibrocystin-1 was a component of the normal focal adhesion complex and that actin and fibrocystin-1 were lost from ARPKD complexes.
Assuntos
Células Epiteliais/fisiologia , Adesões Focais/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Rim Policístico Autossômico Recessivo/patologia , Receptores de Superfície Celular/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/química , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Camundongos , Paxilina/genética , Paxilina/metabolismo , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/fisiopatologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
The regulation of epithelial branching morphogenesis by bone morphogenetic protein-7 depends, in part, on functionally defined cyclic adenosine monophosphate (cAMP)-dependent protein kinases. We previously identified protein kinase-X (PRKX), a cAMP-dependent kinase, as a regulator of epithelial morphogenesis during kidney development and found that it binds to and phosphorylates Polycystin-1. Overexpression of PRKX stimulates renal epithelial cell migration, tubulogenesis, ureteric bud branching, and glomerular induction in embryonic mouse kidney explants in organ cultures. Here we determined the physiological functions of endogenous PRKX. Knockdown by siRNA of PRKX gene expression in a human fetal collecting tubule (HFCT) cell line exceeded 70% and resulted in decreased cell migration and increased adhesion of the cells to a collagen I matrix. In embryonic mouse kidney explants, the same degree of knockdown decreased ureteric bud branching and glomerular induction. Because PRKX BAG-3 PIN-1 and MAGI-1 are all expressed in ureteric bud derivatives, we tested for interactions among them and found that PRKX binds to all three proteins through its WW domain as determined by TransSignal domain arrays, and it coimmunoprecipitated with Pin-1 in HFCT cell lysates. These studies suggest that Polycystin-1 and Pin-1 may mediate the function of PRKX in kidney development.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Rim/citologia , Rim/embriologia , Peptidilprolil Isomerase/metabolismo , Proteínas Quinases/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Adesão Celular/genética , Movimento Celular/genética , Feminino , Camundongos , Morfogênese/genética , Peptidilprolil Isomerase de Interação com NIMA , Técnicas de Cultura de Órgãos , Gravidez , Proteínas Serina-Treonina QuinasesRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a major cause of end-stage renal disease in adults. Autosomal recessive (AR) PKD affects approximately 1:20,000 live-born children with high perinatal mortality. Both diseases have abnormalities in epithelial proliferation, secretion, and cell-matrix interactions, leading to progressive cystic expansion and associated interstitial fibrosis. Cell number in a kidney reflects the balance between proliferation and apoptosis. Apoptosis results from extrinsic (ligand-induced, expression of caspase-8) and intrinsic (mitochondrial damage, expression of caspase-9) triggers. Previous studies have suggested a role for apoptosis in PKD cyst formation and parenchymal destruction. Mechanisms underlying apoptosis in human ADPKD and ARPKD were examined by quantitative immunohistochemistry and Western immunoblot analyses of age-matched normal and PKD tissues. Caspase-8 expression was significantly greater in small cysts and normal-appearing tubules than in larger cysts in ADPKD kidneys. Caspase-8 also appeared early in the disease process of ADPKD. In ARPKD, expression of caspase-8 was most pronounced in later stages of the disease and was not confined to a specific cyst size. In conclusion, apoptosis in human ADPKD is an early event, occurring predominantly in normal-appearing tubules and small cysts, and is triggered by an extrinsic factor, but it occurs later in ARPKD.