RESUMEN
Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.
Asunto(s)
Desarrollo Óseo/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Canales Catiónicos TRPP/fisiología , Animales , Apoptosis , Desarrollo Óseo/fisiología , Diferenciación Celular , Proteína p300 Asociada a E1A/fisiología , Regulación de la Expresión Génica , Genes Reguladores , Células HEK293 , Humanos , Riñón/metabolismo , Modelos Animales , Morfolinos , Osteoblastos/metabolismo , Osteogénesis/fisiología , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/genética , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.
Asunto(s)
Riñón/metabolismo , Potasio en la Dieta/sangre , Receptores de Droga/metabolismo , Simportadores/metabolismo , Administración Oral , Aldosterona/sangre , Animales , Transporte Biológico , Citocromo P-450 CYP11B2/deficiencia , Citocromo P-450 CYP11B2/genética , Canales Epiteliales de Sodio/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Natriuresis , Fosforilación , Potasio en la Dieta/administración & dosificación , Potasio en la Dieta/orina , Receptores de Droga/deficiencia , Receptores de Droga/genética , Miembro 3 de la Familia de Transportadores de Soluto 12 , Simportadores/deficiencia , Simportadores/genética , Factores de Tiempo , Equilibrio HidroelectrolíticoRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.
Asunto(s)
NADP Transhidrogenasa AB-Específica , Riñón Poliquístico Autosómico Dominante , Canales Catiónicos TRPP , Humanos , Animales , Ratones , Modelos Animales de Enfermedad , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/terapia , Riñón/patología , Riñón/fisiología , NADP Transhidrogenasa AB-Específica/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na(+)-K(+)-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman's capsule, the thin and thick limbs of Henle's loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na(+)-K(+)-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.
Asunto(s)
Proteínas Activadoras de GTPasa/análisis , Túbulos Renales Distales/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Riñón/metabolismo , Asa de la Nefrona/metabolismo , Ratones , Ratones EndogámicosRESUMEN
Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) that inactivates 11ß-hydroxy glucocorticoids. High expression levels of 11ß-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11ß-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11ß-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.
Asunto(s)
Túbulos Renales Distales/efectos de los fármacos , Transporte de Proteínas , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Adrenalectomía , Aldosterona/metabolismo , Aldosterona/farmacología , Animales , Especificidad de Anticuerpos , Corticosterona/administración & dosificación , Corticosterona/metabolismo , Corticosterona/farmacología , Túbulos Renales Distales/metabolismo , Masculino , Ratones , Nefronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Glucocorticoides/inmunología , Receptores de Mineralocorticoides/inmunología , Sodio en la Dieta/administración & dosificación , Sodio en la Dieta/farmacologíaRESUMEN
The polycystin-1 and 2 proteins, encoded by the genes mutated in Autosomal Dominant Polycystic Kidney Disease, are connected to a large number of biological pathways. While the nature of these connections and their relevance to the primary functions of the polycystin proteins have yet to be fully elucidated, it is clear that many of them are mediated by or depend upon cleavage of the polycystin-1 protein. Cleavage of polycystin-1 at its G protein coupled receptor proteolytic site is an obligate step in the protein's maturation and in aspects of its trafficking. This cleavage may also serve to prime polycystin-1 to play a role as a non-canonical G protein coupled receptor. Cleavage of the cytoplasmic polycystin-1C terminal tail releases fragments that are able to enter the nucleus and the mitochondria and to influence their activities. Understanding the nature of these cleavages, their regulation and their consequences is likely to provide valuable insights into both the physiological functions served by the polycystin proteins and the pathological consequences of their absence.