RESUMEN
Notch pathway activation in podocytes has been shown to play an important role in diabetic kidney disease (DKD) development; however, the receptors and ligands involved in the process have not been identified. Here, we report that conditional deletion of Notch1 in podocytes using NPHS2(cre)Notch1(flox/flox) animals resulted in marked amelioration of DKD. On the contrary, podocyte-specific genetic deletion of Notch2 had no effect on albuminuria and mesangial expansion. Notch1-null podocytes were protected from apoptosis and dedifferentiation in vitro, likely explaining the protective phenotype in vivo. Deletion of Notch1 in podocytes also resulted in an increase in Notch2 expression, indicating an interaction between the receptors. At the same time, transgenic overexpression of Notch2 in podocytes did not induce phenotypic changes, while constitutive expression of Notch1 caused rapid development of albuminuria and glomerulosclerosis. In summary, our studies indicate that Notch1 plays a distinct (nonredundant) role in podocytes during DKD development.
Asunto(s)
Apoptosis , Desdiferenciación Celular , Nefropatías Diabéticas/metabolismo , Mesangio Glomerular/metabolismo , Podocitos/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular Transformada , Células Cultivadas , Cruzamientos Genéticos , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/prevención & control , Mesangio Glomerular/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Podocitos/patología , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismo , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch2/química , Receptor Notch2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Diabetic kidney disease (DKD) is the single most common cause of albuminuria and end-stage kidney disease in the United States. We found increased expression of Wnt/ß-catenin (Ctnnb1) pathway transcripts and proteins in glomeruli and podocytes of patients and mouse models of DKD. Mice with podocyte-specific expression of stabilized Ctnnb1 exhibited basement membrane abnormalities, albuminuria, and increased susceptibility to glomerular injury. Mice with podocyte-specific deletion of Ctnnb1 or podocyte-specific expression of the canonical Wnt inhibitor Dickkopf-related protein 1 (Dkk1) also showed increased susceptibility to DKD. Podocytes with stabilized Ctnnb1 were less motile and less adhesive to different matrices. Deletion of Ctnnb1 in cultured podocytes increased the expression of podocyte differentiation markers and enhanced cell motility; however, these cells were more susceptible to apoptosis. These results indicate that Wnt/Ctnnb1 signaling in podocytes plays a critical role in integrating cell adhesion, motility, cell death, and differentiation. Balanced Ctnnb1 expression is critical for glomerular filtration barrier maintenance.
Asunto(s)
Diferenciación Celular , Podocitos/citología , Podocitos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Albuminuria/complicaciones , Albuminuria/metabolismo , Animales , Apoptosis/genética , Adhesión Celular/genética , Diferenciación Celular/genética , Supervivencia Celular/genética , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Podocitos/patología , Estabilidad Proteica , Transducción de Señal/genética , Proteínas Wnt/genética , beta Catenina/deficiencia , beta Catenina/genéticaRESUMEN
Chronic kidney disease is a leading cause of death in the United States. Tubulointerstitial fibrosis (TIF) is considered the final common pathway leading to end-stage renal disease (ESRD). Here, we used pharmacologic, genetic, in vivo, and in vitro experiments to show that activation of the Notch pathway in tubular epithelial cells (TECs) in patients and in mouse models of TIF plays a role in TIF development. Expression of Notch in renal TECs was found to be both necessary and sufficient for TIF development. Genetic deletion of the Notch pathway in TECs reduced renal fibrosis. Consistent with this, TEC-specific expression of active Notch1 caused rapid development of TIF. Pharmacologic inhibition of Notch activation using a γ-secretase inhibitor ameliorated TIF. In summary, our experiments establish that epithelial injury and Notch signaling play key roles in fibrosis development and indicate that Notch blockade may be a therapeutic strategy to reduce fibrosis and ESRD development.
Asunto(s)
Células Epiteliales/metabolismo , Túbulos Renales/citología , Riñón/metabolismo , Riñón/patología , Receptor Notch1/metabolismo , Transducción de Señal/fisiología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular , Células Epiteliales/citología , Fibrosis/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/citología , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Nefritis Intersticial/metabolismo , Nefritis Intersticial/patología , Receptor Notch1/genética , Proteínas Serrate-Jagged , Factor de Transcripción HES-1RESUMEN
Recent studies indicate that the Notch signaling pathway plays an important role in the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and activated (cleaved) receptors (Notch1 and Notch2) in healthy human kidneys and in renal biopsies from a wide variety of kidney diseases. These included patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, diabetic kidney disease, and FSGS. We found that cleaved Notch1, Notch2, and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlated with the amount of proteinuria across all disease groups. The degree of glomerulosclerosis correlated with podocyte expression of cleaved Notch1, while the severity of tubulointerstitial fibrosis and the estimated glomerular filtration rate correlated with expression of cleaved Notch1 in the tubulointerstitium. Hence, our results raise the possibility that Notch pathway activation is a common mechanism in the pathophysiology of a wide range of acquired renal diseases.
Asunto(s)
Albuminuria/etiología , Riñón/fisiología , Receptores Notch/fisiología , Transducción de Señal/fisiología , Adulto , Anciano , Femenino , Glomeruloesclerosis Focal y Segmentaria/etiología , Humanos , Inmunohistoquímica , Riñón/patología , Masculino , Persona de Mediana Edad , Receptores Notch/análisisRESUMEN
The incidence of End Stage Renal Disease (ESRD) is approximately 50% higher in men than women. In order to understand the molecular basis of this gender disparity, we examined sex specific gene expression patterns in control and diseased, human and murine kidney samples. Using the Affymetrix platform we performed comprehensive gene expression analysis on 42 microdissected human kidney samples (glomeruli and tubules). We identified 67 genes with gender biased expression in healthy human kidneys and 24 transcripts in diseased male and female human kidneys. Similar analysis performed in mice using male and female control and doxorubicin induced nephrotic syndrome kidneys identified significantly larger number of differentially expressed transcripts. The majority of genes showing gender biased expression either in diseased human and murine kidneys were different from those differentially expressed in healthy kidneys. Only 9 sexually dimorphic transcripts were common to healthy human and murine kidneys and five showed differential regulation in both human and murine diseased kidneys. In humans, sex biased genes showed statistical enrichment only to sex chromosomes while in mice they were enriched to sex chromosomes and various autosomes. Thus we present a comprehensive analysis of gender biased genes in the kidney. We show that sexually dimorphic genes in the kidney show species specific regulation. Our results also indicate that male and female kidneys respond differently to injury. These studies could provide the basis for the development of new treatment strategies for men and women with kidney disease.
Asunto(s)
Regulación de la Expresión Génica , Enfermedades Renales/metabolismo , Riñón/metabolismo , Caracteres Sexuales , Adulto , Albuminuria/inducido químicamente , Albuminuria/genética , Albuminuria/metabolismo , Animales , Doxorrubicina/toxicidad , Femenino , Perfilación de la Expresión Génica , Humanos , Riñón/efectos de los fármacos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/genética , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Fenotipo , ARN Mensajero/biosíntesis , Especificidad de la Especie , Donantes de TejidosRESUMEN
Several gene array studies have suggested that osteopontin (Opn) expression strongly correlates with albuminuria and glomerular disease. Urinary Opn concentration and kidney Opn immunoreactivity were found to be increased in patients with steroid-sensitive nephrotic syndrome. In addition, renal Opn mRNA was increased in the Ins2(Akita) mouse model of type 1 diabetic nephropathy, in the LPS-induced albuminuria model, and in glomeruli of puromycin aminonucleotide-induced nephrotic rats. Opn knockout mice did not develop albuminuria in response to LPS injection, and Opn knockout mice were protected from diabetes-induced albuminuria and mesangial expansion. In the glomerulus, Opn immunostaining was increased specifically in podocytes. Treatment of podocytes with recombinant Opn activated the NF-kappaB pathway, increased expression of urokinase plasminogen activator and matrix metalloproteinases 2 and 9, and increased podocyte motility. Taken together, these results indicate that Opn plays an important role in the development of albuminuria, possibly by modulating podocyte signaling and motility.
Asunto(s)
Albuminuria/metabolismo , Albuminuria/fisiopatología , Osteopontina/genética , Osteopontina/metabolismo , Albuminuria/patología , Animales , Biopsia , Células Cultivadas , Niño , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Síndrome Nefrótico/fisiopatología , Osteopontina/farmacología , Fenotipo , Podocitos/efectos de los fármacos , Podocitos/patología , Podocitos/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiologíaRESUMEN
Albuminuria associated with sclerosis of the glomerulus leads to a progressive decline in renal function affecting millions of people. Here we report that activation of the Notch pathway, which is critical in glomerular patterning, contributes to the development of glomerular disease. Expression of the intracellular domain of Notch1 (ICN1) was increased in glomerular epithelial cells in diabetic nephropathy and in focal segmental glomerulosclerosis. Conditional re-expression of ICN1 in vivo exclusively in podocytes caused proteinuria and glomerulosclerosis. In vitro and in vivo studies showed that ICN1 induced apoptosis of podocytes through the activation of p53. Genetic deletion of a Notch transcriptional partner (Rbpj) specifically in podocytes or pharmacological inhibition of the Notch pathway (with a gamma-secretase inhibitor) protected rats with proteinuric kidney diseases. Collectively, our observations suggest that Notch activation in mature podocytes is a new mechanism in the pathogenesis of glomerular disease and thus could represent a new therapeutic target.