RESUMEN
AIM: The Na(+) /H(+) exchanger NHE3 activity decreases in the proximal tubule of spontaneously hypertensive rats (SHRs) as blood pressure increases, and this reduction is correlated with higher NHE3 phosphorylation levels at the PKA consensus site serine 552. This study tested the hypothesis that this lowered NHE3 activity is associated with an increase in PKA activity and expression, and/or a decrease in protein phosphatase-1 (PP1) activity and expression. METHODS: Proximal tubule NHE3 activity was measured as the rate of bicarbonate reabsorption by stationary microperfusion. NHE3 phosphorylation and protein expression were determined by immunoblotting. PKA and PP1 activities were determined using specific substrates under optimal enzymatic conditions. RESULTS: The PKA activator, 6-MB-cAMP, increased the phosphorylation levels of NHE3 at serine 552 in the renal cortex; this increase happens to a much greater extent in young pre-hypertensive SHRs (Y-SHRs) compared to adult SHRs with established hypertension (A-SHRs). Likewise, the inhibitory effect of 6-MB-cAMP on NHE3 transport activity was much more pronounced in the proximal tubules of Y-SHRs than in those of A-SHRs. Renal cortical activity of PKA was not significantly different between Y-SHRs and A-SHRs. On the other hand, Y-SHRs exhibited higher protein phosphatase 1 (PP1) activity, and their expression of the PP1 catalytic subunit PP1α in the renal cortex was also higher than in A-SHRs. CONCLUSION: Collectively, these results support the idea that the lower NHE3 transport activity and higher phosphorylation occurring after the development of hypertension in SHRs are due, at least in part, to reduced PP1-mediated dephosphorylation of NHE3 at serine 552.
Asunto(s)
Hipertensión/metabolismo , Proteína Fosfatasa 1/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Immunoblotting , Túbulos Renales Proximales/metabolismo , Masculino , Fosforilación , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Intercambiador 3 de Sodio-HidrógenoRESUMEN
Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.
Asunto(s)
Angiotensina II/farmacología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/metabolismo , Cloruro de Amonio/farmacología , Androstadienos/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Tampones (Química) , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Genisteína/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Imidazoles/farmacología , Túbulos Renales Proximales/enzimología , Losartán/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Piridinas/farmacología , Ratas , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , ATPasas de Translocación de Protón Vacuolares/genética , Wortmanina , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Túbulos Renales Proximales/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/ultraestructura , Microscopía Electrónica , Microvellosidades/enzimología , Microvellosidades/metabolismo , Pruebas de Precipitina , Unión Proteica , Conejos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/inmunologíaRESUMEN
BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS: Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.