RESUMO
Background Renal glomeruli are the primary target of injury in diabetic nephropathy (DN), and the glomerular podocyte has a key role in disease progression.Methods To identify potential novel therapeutic targets for DN, we performed high-throughput molecular profiling of G protein-coupled receptors (GPCRs) using human glomeruli.Results We identified an orphan GPCR, Gprc5a, as a highly podocyte-specific gene, the expression of which was significantly downregulated in glomeruli of patients with DN compared with those without DN. Inactivation of Gprc5a in mice resulted in thickening of the glomerular basement membrane and activation of mesangial cells, which are two hallmark features of DN in humans. Compared with wild-type mice, Gprc5a-deficient animals demonstrated increased albuminuria and more severe histologic changes after induction of diabetes with streptozotocin. Mechanistically, Gprc5a modulated TGF-ß signaling and activation of the EGF receptor in cultured podocytes.Conclusions Gprc5a has an important role in the pathogenesis of DN, and further study of the podocyte-specific signaling activity of this protein is warranted.
Assuntos
Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Podócitos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Actinas/metabolismo , Albuminúria/etiologia , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Regulação para Baixo , Receptores ErbB/metabolismo , Fibronectinas/metabolismo , Membrana Basal Glomerular/patologia , Humanos , Células Mesangiais , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.
Assuntos
Barreira de Filtração Glomerular/patologia , Glomerulonefrite/patologia , Microscopia Confocal , Microscopia de Fluorescência , Expansão de Tecido/métodos , Animais , Autoanticorpos , Biomarcadores/metabolismo , Colágeno Tipo IV/imunologia , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Barreira de Filtração Glomerular/imunologia , Barreira de Filtração Glomerular/metabolismo , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , RatosRESUMO
Using a convenience sampling, nurse educators representing 10 countries were surveyed to describe required clinical education for advanced practice beyond basic traditional nursing education. This article explores the many factors currently influencing the structure and diversity of these clinical experiences worldwide.