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1.
Lancet ; 358(9287): 1102, 2001 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-11594329
2.
Kidney Int ; 60(3): 1182-96, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11532115

RESUMO

BACKGROUND: Patients undergoing successful kidney transplantation often manifest overt hypophosphatemia associated with exaggerated phosphaturia during the early post-transplant period (2 weeks to 3 months). The mechanism for this phenomenon has not been fully elucidated. We tested the hypothesis that a circulating serum factor [non-parathyroid hormone (non-PTH)], which operates during chronic renal failure (CRF) to maintain phosphate (Pi) homeostasis, can increase fractional excretion of Pi (FE(PO4)) in normal functioning kidney grafts during the early post-transplant period, thereby causing phosphaturia and hypophosphatemia. METHODS: Five groups of patients were studied: control subjects (group 1, N = 16), "early" (2 weeks to 1 month) post-transplant patients (group 2, N = 22), "late" (9 to 12 months) post-transplant patients (group 3, N = 14), patients with advanced CRF (glomerular filtration rate = 30 to 40 mL/min; group 4, N = 8), and patients who suffered from end-stage renal failure and were treated by chronic hemodialysis (group 5, N = 14). Group 2 manifested significant hypophosphatemia and phosphaturia when compared with groups 1 and 3 (Pi = 0.9 +/- 0.003 mg/dL, FE(PO4) = 68+/- 5%, P < 0.0005 vs. groups 1 and 3). Sera were taken from each of the five subject groups and applied to the proximal tubular opossum kidney (OK) cells. The activity of Na/Pi-type 4 (that is, OK-specific type II transporter) was evaluated by measuring Na(+)-dependent (32)Pi flux. The expression of Na/Pi type II mRNA and the abundance of Na/Pi protein were determined by Northern and Western blot assays, respectively. RESULTS: When compared with sera from groups 1 and 3, 10% sera taken from groups 2, 4, and 5 (incubated overnight with OK cells) inhibited (32)Pi flux by 25 to 30% (P < 0.0003). Both Na/Pi mRNA and the expression of Na/Pi protein were markedly augmented under the same conditions (P < 0.05 groups 2, 4, and 5 vs. groups 1 and 3). Time-course analysis revealed that the up-regulation of Na/Pi protein by sera from groups 2, 4, and 5 was observed as early as four hours of incubation, whereas augmented abundance of Na/Pi mRNA was only seen after eight hours of incubation. The addition of PTH (1-34) to sera from groups 2, 4, and 5 abolished the augmented expression of NaPi protein. We labeled OK cell surface membrane proteins with N-hydroxysuccinimide bound to biotin (NHS-SS-biotin). Biotinylated transporters incubated with the different sera were precipitated by strepavidin and identified by Western blot analysis. Cells incubated in sera from group 2 showed increased membrane bound transporter when compared with control sera, whereas the intracellular pool of the transporter was comparable between the two groups. CONCLUSION: A non-PTH circulating serum factor (possibly phosphatonin) that increases FE(PO4) during CRF is also responsible for phosphaturia and hypophosphatemia in the early period following successful kidney transplantation. The putative factor inactivates Na/Pi activity along with inhibition of the transporter trafficking from the cell membrane into the cytosol.


Assuntos
Hipofosfatemia/etiologia , Transplante de Rim , Complicações Pós-Operatórias , Simportadores , Adulto , Idoso , Animais , Transporte Biológico , Sangue , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Feminino , Humanos , Hipofosfatemia/sangue , Hipofosfatemia/urina , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Gambás , Radioisótopos de Fósforo , RNA Mensageiro/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II , Fator Tímico Circulante/análise
3.
Am J Physiol Renal Physiol ; 281(3): F428-33, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11502592

RESUMO

Acute administration of dihydroxycholecalciferol [1,25(OH)(2)D(3)] blunts phosphaturia and increases urinary cAMP excretion in parathyroid hormone (PTH)-infused parathyroidectomized (PTX) rats. Because chronic administration of 1,25(OH)(2)D(3) enhances the phosphaturic response to exogenous parathyroid hormone despite blunting of urinary cAMP excretion, we have examined the expression of the renal cortex type II Na-P(i) cotransporter (NaPi-2) mRNA and protein in 1) chronic PTX Sabra rats, 2) PTX rats receiving a physiological dose of 1,25(OH)-2-D(3), 3) PTX rats receiving 35 ng/h of PTH, and 4) rats receiving both PTH and 1,25(OH)(2)D(3), for 7 days via osmotic minipumps. Our results confirm that there is increased phosphaturia in the PTH+1,25(OH)(2)D(3)-infused animals despite blunting of urinary cAMP excretion, a reduced filtered load of phosphate, and lack of a phosphaturic effect by 1,25(OH)(2)D(3) alone. Both PTH and 1,25(OH)(2)D(3) significantly reduced expression of renal cortex NaPi-2 mRNA and NaPi-2 protein, and the administration of PTH together with 1,25(OH)(2)D(3) had additive effects in further decreasing NaPi-2 mRNA and NaPi-2 protein levels. Expression of two other epithelial transporters, type 1 Na-sulfate and type 1 Na-glucose cotransporters, were not different between the groups, suggesting specificity of the effects of PTH and 1,25(OH)(2)D(3) on phosphate transport. The effect of chronic administration of 1,25(OH)(2)D(3) has not been noted previously, and the cellular mechanisms and signaling processes that mediate the decrease in NaPi-2 remain to be determined.


Assuntos
Calcitriol/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Rim/fisiologia , Fosfatos/metabolismo , Simportadores , Teriparatida/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Northern Blotting , AMP Cíclico/urina , Regulação da Expressão Gênica/efeitos dos fármacos , Infusões Parenterais , Rim/efeitos dos fármacos , Córtex Renal/fisiologia , Masculino , Paratireoidectomia , Fosfatos/sangue , Fosfatos/urina , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/genética , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II , Teriparatida/administração & dosagem , Teriparatida/antagonistas & inibidores
4.
Kidney Int ; 60(2): 694-704, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11473652

RESUMO

BACKGROUND: Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. METHODS: K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. RESULTS: K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V(max) of brush-border membrane (BBM) Na/Pi cotransport activity (1943 +/- 95 in control vs. 1184 +/- 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 1152 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. CONCLUSION: K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.


Assuntos
Proteínas de Transporte/metabolismo , Glucosilceramidas/metabolismo , Túbulos Renais Proximais/metabolismo , Fluidez de Membrana/fisiologia , Fosfatos/metabolismo , Deficiência de Potássio/metabolismo , Simportadores , Animais , Proteínas de Transporte/genética , Gangliosídeo G(M3)/metabolismo , Expressão Gênica/fisiologia , Hipopotassemia/metabolismo , Cinética , Masculino , Microvilosidades/metabolismo , Oócitos/metabolismo , Fósforo/urina , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Xenopus laevis
5.
J Clin Invest ; 104(4): 483-94, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10449440

RESUMO

Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.


Assuntos
Proteínas de Transporte/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Microtúbulos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Simportadores , Animais , Colchicina/farmacologia , Endocitose/efeitos dos fármacos , Imuno-Histoquímica , Túbulos Renais Proximais/ultraestrutura , Masculino , Microscopia Eletrônica , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Paclitaxel/farmacologia , Fosfatos/metabolismo , Ratos , Ratos Wistar , Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa
6.
Am J Physiol ; 276(6): C1398-404, 1999 06.
Artigo em Inglês | MEDLINE | ID: mdl-10362603

RESUMO

Recently, we cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (Si) transport protein (Na-Si cotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-Si cotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of Si, which was associated with a 2.4-fold decrease in BBM Na-Si cotransport activity. The decrease in Na-Si cotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Si cotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-Si cotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Si reabsorption by causing decreases in BBM Na-Si cotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-Si cotransport activity during MA.


Assuntos
Acidose/metabolismo , Cloreto de Amônio/administração & dosagem , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Rim/metabolismo , Simportadores , Cloreto de Amônio/farmacologia , Animais , Artérias , Bicarbonatos/sangue , Fenômenos Fisiológicos Sanguíneos , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Dieta , Concentração de Íons de Hidrogênio , Masculino , Microvilosidades/metabolismo , Concentração Osmolar , RNA Mensageiro/fisiologia , Ratos , Ratos Sprague-Dawley , Cotransportador de Sódio-Sulfato , Sulfatos/urina
7.
Am J Physiol ; 276(1): F72-8, 1999 01.
Artigo em Inglês | MEDLINE | ID: mdl-9887082

RESUMO

In the present study, we determined the effect of epidermal growth factor (EGF; 10 microgram/100 g body wt) on sodium gradient-dependent phosphate transport (Na-Pi cotransport) regulation in suckling (12-day-old) and weaned (24-day-old) rats. Weaned rats had higher proximal tubular brush border membrane vesicle (BBMV) Na-Pi cotransport activity (232 +/- 16 in weaned vs. 130 +/- 9 pmol. 10 s-1. mg protein-1 in suckling rats, P < 0.05). Chronic treatment with EGF induced inhibition of BBMV Na-Pi cotransport in both suckling (130 +/- 9 vs. 104 +/- 7 pmol. 10 s-1. mg protein-1, P < 0. 05) and weaned rats (232 +/- 16 vs. 145 +/- 9 pmol. 10 s-1. mg protein-1, P < 0.005). The inhibitory effect was selective for Na-Pi cotransport as there was no inhibition of Na-glucose cotransport. Weaned rats had a higher abundance of BBMV NaPi-2 protein than suckling rats (increase of 54%, P < 0.001) and a twofold increase in NaPi-2 mRNA. The EGF-induced inhibition of Na-Pi transport was paralleled by decreases in NaPi-2 protein abundance in both weaned (decrease of 26%, P < 0.01) and suckling (decrease of 27%, P < 0.01) animals. In contrast, there were no changes in NaPi-2 mRNA abundance. We conclude that proximal tubule BBMV Na-Pi cotransport activity, NaPi-2 protein abundance, and NaPi-2 mRNA abundance are higher in weaned than in suckling rats. EGF inhibits Na-Pi cotransport activity in BBMV isolated from suckling and weaned rats, and this inhibition is mediated via a decrease in NaPi-2 protein abundance, in the absence of a change in NaPi-2 mRNA.


Assuntos
Animais Lactentes/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Fator de Crescimento Epidérmico/farmacologia , Simportadores , Desmame , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Proximais/metabolismo , Microvilosidades/metabolismo , Fosfatos/urina , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Cotransportadoras de Sódio-Fosfato
8.
Kidney Int ; 53(5): 1288-98, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9573544

RESUMO

Metabolic acidosis results in impaired renal tubular phosphate reabsorption and proximal tubular apical brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na/Pi cotransport) activity. In the present study we investigated the cellular mechanisms responsible for decreased Na/Pi cotransport activity following six hours to 10 days of metabolic acidosis induced by ingestion of NH4Cl. Urinary Pi excretion was significantly increased and BBM Na/Pi cotransport activity was progressively and significantly decreased by 18% at six hours, 24% at 12 hours, 32% at 24 hours, and 61% after 10 days of metabolic acidosis. The progressive and time-dependent decreases in BBM cotransport activity were associated with progressive decreases in BBM NaPi-2 protein (43% at 12 hr, 54% at 24 hr and 66% at 10 days) and cortical NaPi-2 mRNA (22% at 12 hr, 54% at 24 hr and 56% at 10 days) abundance. Interestingly, following six hours of metabolic acidosis, there was a significant 29% decrease in BBM NaPi-2 protein abundance that was not associated with decreases in either cortical homogenate NaPi-2 protein or cortical NaPi-2 mRNA abundance. In additional studies we found that the effects of chronic metabolic acidosis on Na/Pi cotransport activity were independent of endogenous parathyroid hormone activity, but were somewhat dependent on dietary Pi intake. In rats fed a high or a normal Pi diet metabolic acidosis caused significant decreases in Na/Pi cotransport activity, NaPi-2 protein and NaPi-2 mRNA abundance, however, in rats fed a low Pi diet the inhibitory effect of metabolic acidosis on Na/Pi cotransport were minimal and not significant. These results indicate that in chronic (> or = 12 hr) metabolic acidosis the progressive decrease in BBM Na/Pi cotransport activity is most likely mediated by decrease in BBM NaPi-2 protein and cortical mRNA abundance. In contrast, in acute (< or = 6 hr) metabolic acidosis the decrease in BBM Na/Pi cotransport activity is likely mediated by changes in the trafficking of the NaPi-2 protein that is, enhanced internalization from and/or impaired delivery of the NaPi-2 protein to the apical BBM.


Assuntos
Acidose/metabolismo , Rim/metabolismo , Fosfatos/metabolismo , Simportadores , Doença Aguda , Animais , Bicarbonatos/sangue , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Doença Crônica , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Transporte de Íons , Córtex Renal/metabolismo , Masculino , Lipídeos de Membrana/metabolismo , Microvilosidades/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatos/urina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Cotransportadoras de Sódio-Fosfato
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