RESUMEN
Calcineurin is an important regulator of extracellular matrix (ECM) accumulation in the kidney but functions in a cell-specific manner. Previously, we identified a novel role for calcineurin in mesangial cells where calcineurin activity is required for TGFbeta-mediated induction of fibronectin expression. In this study, we examined the role of the calcineurin substrate NFATc in transcriptional regulation of fibronectin. First, inhibition of calcineurin blocks TGFbeta induction of the fibronectin promoter. Moreover, expression of constitutively active calcineurin in mesangial cells is sufficient to increase fibronectin transcription. Next, inhibition of the calcineurin substrate NFATc1 blocked TGFbeta-mediated activation of the fibronectin promoter. Finally, stable expression of a dominant-negative NFATc protein reduced transcriptional activation of the promoter and inhibited TGFbeta-mediated fibronectin expression. In conclusion, TGFbeta activation of calcineurin in mesangial cells results in regulation of ECM accumulation at least in part by direct transcriptional activity of NFATc on the fibronectin promoter.
Asunto(s)
Fibronectinas/metabolismo , Factores de Transcripción NFATC/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Línea Celular , Células Cultivadas , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Factores de Transcripción NFATC/genética , Unión Proteica/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tacrolimus/farmacología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND: Use of calcineurin inhibitors is frequently limited by fibrosis, closely linked with increased transforming growth factor (TGF)-beta. However, mechanisms of extracellular matrix expansion and TGFbeta regulation following calcineurin inhibition are unknown. Mice lacking specific calcineurin catalytic subunit isoforms may offer important insight into this pathway. METHODS: We compared mice lacking the alpha or beta isoform to a model of cyclosporin nephrotoxicity. Histological features common with cyclosporin nephrotoxicity including matrix expansion, arteriole hyalinization, and inflammation were assessed. Next, regulation specifically of fibronectin and TGFbeta was examined in vivo and in vitro. Finally, the role of TGFbeta in upregulation of fibronectin with loss of calcineurin activity was examined. RESULTS: Loss of the alpha isoform results in histologic features and matrix expansion similar to cyclosporin, whereas loss of the beta does not. Fibronectin and TGFbeta are increased and renal function is impaired in alpha-null and aged alpha+/-. In primary alpha-/- renal fibroblasts, nuclear translocation of the calcineurin substrate NFATc is normal but regulation is lost in beta-null fibroblasts, confirming that the isoforms have distinct functions. Consistent with in vivo findings, alpha-null cells have increased fibronectin and TGFbeta. However, neutralizing TGFbeta antibody did not reduce fibronectin accumulation. CONCLUSIONS: Our data show that calcineurin-alpha is key to regulation of fibrosis and TGFbeta and loss of this isoform reproduces features of cyclosporine nephrotoxicity in vivo and in vitro. In addition, we show that upregulation of TGFbeta and fibronectin likely result from a shared mechanism, but changes in fibronectin expression are independent of TGFbeta in renal fibroblasts.
Asunto(s)
Calcineurina/deficiencia , Calcineurina/metabolismo , Regulación de la Expresión Génica , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Calcineurina/genética , Células Cultivadas , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Enfermedades Renales/genética , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Hyperphosphatemia is thought to underlie medial vascular calcification in advanced renal failure, but calcification can occur in other conditions in the absence of hyperphosphatemia, indicating that additional factors are important. To identify these factors, a model of medial calcification in rat aorta in vitro was developed. Aortic rings from rats were incubated in serum-free medium for 9 d, and calcification was measured as incorporation of (45)Ca and confirmed by histology and x-ray diffraction. No calcification occurred in normal vessels despite elevated free Ca(2+) and PO(4)(3-) concentrations of 1.8 mM and 3.8 mM, respectively, but mechanical injury resulted in extensive calcification in the media. Co-incubation studies revealed that normal aortas produced a soluble inhibitor of calcification in injured vessels that was destroyed by alkaline phosphatase. Culture of normal aortas with alkaline phosphatase resulted in calcification of the elastic lamina identified as hydroxyapatite by x-ray diffraction. This effect of alkaline phosphatase was not due to dephosphorylation of osteopontin (OPN), and calcification was not increased in aortas from OPN-deficient mice. The inhibitor was identified as pyrophosphate on the basis of the calcification induced in aortas cultured with inorganic pyrophosphatase, the inhibition of calcification in injured aortas by pyrophosphate, and the production of inhibitory levels of pyrophosphate by normal aortas. No calcification occurred under any conditions at a normal PO(4)(3-) concentration. It is concluded that elevated concentrations of Ca(2+) and PO(4)(3-) are not sufficient for medial vascular calcification because of inhibition by pyrophosphate. Alkaline phosphatase can promote calcification by hydrolyzing pyrophosphate, but OPN is not an endogenous inhibitor of calcification in rat aorta.
Asunto(s)
Calcio/metabolismo , Difosfatos/metabolismo , Fosfatos/metabolismo , Sialoglicoproteínas/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Aorta/patología , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero/farmacología , Difosfatos/química , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Transgénicos , Osteopontina , Fenotipo , Ratas , Ratas Sprague-Dawley , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Factores de Tiempo , Difracción de Rayos XRESUMEN
The Na-K-2Cl cotransporter (NKCC1) is one of several transporters that have been linked to hypertension, and its inhibition reduces vascular smooth muscle tone and blood pressure. NKCC1 in the rat aorta is stimulated by vasoconstrictors and inhibited by nitrovasodilators, and this is linked to the contractile state of the smooth muscle. To determine whether blood pressure also regulates NKCC1, we examined the acute effect of hypertension on NKCC1 in rats after aortic coarctation. In the hypertensive aorta (28-mmHg rise in mean blood pressure), an increase in NKCC1 activity (measured as bumetanide-sensitive (86)Rb efflux) was apparent by 16 h and reached a plateau of 62% greater than control at 48 h. In contrast, there was a slight decrease in NKCC1 activity in the hypotensive aorta (21% decrease in mean blood pressure). Measurement of NKCC1 mRNA by real-time PCR revealed a fivefold increase in the hypertensive aorta compared with the hypotensive aorta or sham aorta. The inhibition by bumetanide of isometric force response to phenylephrine was significantly greater in the hypertensive aorta than in the control aorta or hypotensive aorta. We conclude that NKCC1 in rat aortic smooth muscle is regulated by blood pressure, most likely through changes in transporter abundance. This upregulation of NKCC1 is associated with a greater contribution to force generation in the hypertensive aorta. This is the first demonstration that NKCC1 in vascular smooth muscle is regulated by blood pressure and indicates that this transporter is important in the acute response of vascular smooth muscle to hypertension.
Asunto(s)
Presión Sanguínea/fisiología , Músculo Liso Vascular/fisiología , Simportadores de Cloruro de Sodio-Potasio/fisiología , Animales , Coartación Aórtica/fisiopatología , Bumetanida/farmacología , Cardiomegalia/fisiopatología , Diuréticos/farmacología , Inhibidores Enzimáticos/farmacología , Hipertensión/fisiopatología , Contracción Isométrica/fisiología , Masculino , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores de Cloruro de Sodio-Potasio/biosíntesis , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
Aldosterone increases cation transport and contractility of vascular smooth muscle, but the specific transporter involved and how it is linked to smooth muscle tone is unknown. Because the Na-K-2Cl cotransporter (NKCC1) contributes to vascular smooth muscle contraction and is regulated by vasoactive compounds, we sought to determine whether this transporter is a target of aldosterone in rat aorta. Treatment of adrenalectomized rats with aldosterone for 7 days resulted in a 63% increase in NKCC1 activity as measured by bumetanide-sensitive efflux of 86Rb+. Treatment of normal aortas in culture with aldosterone for 3 and 7 days resulted in 29% and 47% increases in NKCC1 activity, respectively. Aldosterone had no acute effect on 86Rb+ efflux. Stimulation of NKCC1 was blocked by spironolactone, a mineralocorticoid receptor antagonist, but not by RU38486, a glucocorticoid receptor antagonist. Aldosterone did not augment the stimulation of NKCC1 by phenylephrine and did not increase NKCC1 mRNA as determined by real-time polymerase chain reaction. We conclude that aldosterone regulates the Na-K-2Cl cotransporter in vascular smooth muscle through classic mineralocorticoid receptors but not through changes in the abundance of NKCC1 mRNA. This could account for the increase in Na+, K+, and Cl- fluxes previously observed in vascular smooth muscle from mineralocorticoid-treated animals and may contribute to increased vascular tone.