RESUMEN
Phosphate (Pi) homeostasis is regulated by renal, intestinal, and endocrine mechanisms through which Pi intake stimulates parathyroid hormone (PTH) and fibroblast growth factor-23 secretion, increasing phosphaturia. Mechanisms underlying the early adaptive phase and the role of the intestine, however, remain ill defined. We investigated mineral, endocrine, and renal responses during the first 4 hours after intravenous and intragastric Pi loading in rats. Intravenous Pi loading (0.5 mmol) caused a transient rise in plasma Pi levels and creatinine clearance and an increase in phosphaturia within 10 minutes. Plasma calcium levels fell and PTH levels increased within 10 minutes and remained low or high, respectively. Fibroblast growth factor-23, 1,25-(OH)2-vitamin D3, and insulin concentrations did not respond, but plasma dopamine levels increased by 4 hours. In comparison, gastric Pi loading elicited similar but delayed phosphaturia and endocrine responses but did not affect plasma mineral levels. Either intravenous or gastric loading led to decreased expression and activity of renal Pi transporters after 4 hours. In parathyroidectomized rats, however, only intravenous Pi loading caused phosphaturia, which was blunted and transient compared with that in intact rats. Intravenous but not gastric Pi loading in parathyroidectomized rats also led to higher creatinine clearance and lower plasma calcium levels but did not reduce the expression or activity of Pi transporters. This evidence suggests that an intravenous or intestinal Pi bolus causes rapid phosphaturia through mechanisms requiring PTH and downregulation of renal Pi transporters but does not support a role of the intestine in stimulating renal clearance of Pi.
Asunto(s)
Adaptación Fisiológica , Hormona Paratiroidea/fisiología , Fosfatos/administración & dosificación , Fosfatos/metabolismo , Administración Intravenosa , Administración Oral , Animales , Hipofosfatemia Familiar/etiología , Mucosa Intestinal/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Plasma phosphate concentration is maintained within a relatively narrow range by control of renal reabsorption of filtered inorganic phosphate (P(i)). P(i) reabsorption is a transcellular process that occurs along the proximal tubule. P(i) flux at the apical (luminal) brush border membrane represents the rate-limiting step and is mediated by three Na(+)-dependent P(i) cotransporters (members of the SLC34 and SLC20 families). The putative proteins responsible for basolateral P(i) flux have not been identified. The transport mechanism of the two kidney-specific SLC34 proteins (NaPi-IIa and NaPi-IIc) and of the ubiquitously expressed SLC20 protein (PiT-2) has been studied by heterologous expression to reveal important differences in kinetics, stoichiometry, and substrate specificity. Studies on the regulation of the abundance of the respective proteins highlight significant differences in the temporal responses to various hormonal and nonhormonal factors that can influence P(i) homeostasis. The phenotypes of mice deficient in NaPi-IIa and NaPi-IIc indicate that NaPi-IIa is responsible for most P(i) renal reabsorption. In contrast, in the human kidney, NaPi-IIc appears to have a relatively greater role. The physiological relevance of PiT-2 to P(i) reabsorption remains to be elucidated.
Asunto(s)
Homeostasis/fisiología , Túbulos Renales Proximales/fisiología , Proteínas de Transporte de Fosfato/fisiología , Animales , Humanos , Ratones , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/fisiologíaRESUMEN
BACKGROUND/AIMS: Renal reabsorption of inorganic phosphate (Pi) is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi itself also regulates the expression of cotransporters and the concentration of Pi-regulating hormones, though the signaling pathways are largely unknown. Here, we explored the mechanisms that allow renal proximal cells to adapt to changes in the concentration of Pi. METHODS: opossum kidney (OK) cells, a model of proximal epithelia, were incubated with different concentrations of Pi in the absence/presence of phosphonoformic acid (PFA), a Pi-analogue and SLC34-inhibitor, and of inhibitors of kinases involved in hormonal control of Pi-homeostasis; cells cultured in normal media were treated with uncouplers of oxidative phosphorylation. Then, the intracellular concentration of ATP and/or the Pi-transport capacity of the cultures were analyzed. RESULTS: luminal Pi regulates the Pi-transport and the intracellular ATP levels. Changes in ATP seem secondary to alterations in Pi-transport, rather than ATP acting as a signal. Adaptation of Pi-transport to high Pi was not mimicked by PFA. Transport adaptation was blocked by PFA but not by kinase inhibitors. CONCLUSIONS: in OK cells, adaptation of Pi-transport to luminal Pi does not depend on the same signaling pathways involved in hormonal regulation.
Asunto(s)
Riñón/citología , Fosfatos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Foscarnet/antagonistas & inhibidores , Túbulos Renales Proximales/citología , Zarigüeyas , Fosfatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/antagonistas & inhibidores , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II/metabolismoRESUMEN
The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.
Asunto(s)
Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Animales , Factor-23 de Crecimiento de Fibroblastos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Túbulos Renales/metabolismo , Túbulos Renales/fisiología , Fosfatos/deficiencia , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genéticaRESUMEN
Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)2D3 formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)2D3 stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)2D3 formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)2D3 concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)2D3 and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)2D3, and phosphate concentrations and thus possibly affecting tissue calcification.
Asunto(s)
Calcio/metabolismo , Riñón/efectos de los fármacos , Litio/farmacología , Fosfatos/metabolismo , Animales , Calcitriol/sangre , Calcio/sangre , Calcio/orina , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Glucuronidasa/genética , Glucuronidasa/metabolismo , Riñón/metabolismo , Riñón/fisiología , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Fosfatos/sangre , Fosfatos/orinaRESUMEN
The proximal renal epithelia express three different Na-dependent inorganic phosphate (Pi) cotransporters: NaPi-IIa/SLC34A1, NaPi-IIc/SLC34A3, and PiT2/SLC20A2. Constitutive mouse knockout models of NaPi-IIa and NaPi-IIc suggested that NaPi-IIa mediates the bulk of renal reabsorption of Pi whereas the contribution of NaPi-IIc to this process is minor and probably restricted to young mice. However, many reports indicate that mutations of NaPi-IIc in humans lead to hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report the generation of a kidney-specific and inducible NaPi-IIc-deficient mouse model based on the loxP-Cre system. We found that the specific removal of the cotransporter from the kidneys of young mice does not impair the capacity of the renal epithelia to transport Pi. Moreover, the levels of Pi in plasma and urine as well as the circulating levels of parathyroid hormone, FGF-23, and vitamin D3 remained unchanged. These findings are in agreement with the data obtained with the constitutive knockout model and suggest that, under steady-state conditions of normal dietary Pi, NaPi-IIc is not an essential Na-Pi cotransporter in murine kidneys. However, and unlike the constitutive mutants, the kidney-specific depletion of NaPi-IIc does not result in alteration of the homeostasis of calcium. This suggests that the calcium-related phenotype observed in constitutive knockout mice may not be related to inactivation of the cotransporter in kidney.
Asunto(s)
Calcio/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Animales , Calcitriol/metabolismo , Doxiciclina/farmacología , Raquitismo Hipofosfatémico Familiar/fisiopatología , Factor-23 de Crecimiento de Fibroblastos , Homeostasis/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/deficienciaRESUMEN
Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/metabolismo , Enfermedades Renales Poliquísticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biomarcadores/sangre , Calcitriol/metabolismo , Calcio/sangre , Creatinina/sangre , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Glucuronidasa/metabolismo , Riñón/patología , Proteínas Klotho , Masculino , Ratones Noqueados , Hormona Paratiroidea/sangre , Fosfatos/sangre , Fosforilación , Enfermedades Renales Poliquísticas/sangre , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética , Regulación hacia Arriba , Urea/sangreRESUMEN
UNLABELLED: Renal control of systemic phosphate homeostasis is critical as evident from inborn and acquired diseases causing renal phosphate wasting. At least three transport proteins are responsible for renal phosphate reabsorption: NAPI-IIa (SLC34A1), NAPI-IIc (SLC34A3) and PIT-2 (SLC20A2). These transporters are highly regulated by various cellular mechanisms and factors including acid-base status, electrolyte balance and hormones such as dopamine, glucocorticoids, growth factors, vitamin D3, parathyroid hormone and fibroblast growth factor 23 (FGF23). Whether renal phosphate wasting is caused by inactivating mutations in the NAPI-IIa transporter is controversial. Mutations in the NAPI-IIc transporter cause hereditary hypophosphatemic rickets with hypercalciuria. Besides the primary inherited defects, there are also inherited defects in major regulators of phosphate homeostasis that lead to alterations in phosphate handling. Autosomal dominant hypophosphatemic rickets is due to FGF23 mutations leading to resistance against its own degradation. Similarly, inactivating mutations in the PHEX gene, which causes FGF23 inactivation, cause X-linked hypophosphatemia due to renal phosphate losses. In contrast, mutations in galactosamine:polypeptide N-acetyl-galactosaminyltransferase, responsible for O-glycosylation of FGF23, or in klotho, a cofactor for FGF23 signalling result in hyperphosphatemia. Acquired syndromes of renal phosphate wasting, hypophosphatemia and osteomalacia (tumour-associated osteomalacia) can be due to the excessive synthesis or release of phosphaturic factors (FGF23, FGF-7, MEPE and sFRP4) from mesenchymal tumours. KEYWORDS: bone, FGF23, kidney, phosphate, PTH.
Asunto(s)
Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad , Enfermedades Renales/genética , Fosfatos/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/terapiaRESUMEN
Renal reabsorption of inorganic phosphate (Pi) is mediated by the phosphate transporters NaPi-IIa, NaPi-IIc, and Pit-2 in the proximal tubule brush border membrane (BBM). Dietary Pi intake regulates these transporters; however, the contribution of the specific isoforms to the rapid and slow phase is not fully clarified. Moreover, the regulation of PTH and FGF23, two major phosphaturic hormones, during the adaptive phase has not been correlated. C57/BL6 and NaPi-IIa(-/-) mice received 5 days either 1.2 % (HPD) or 0.1 % (LPD) Pi-containing diets. Thereafter, some mice were acutely switched to LPD or HPD. Plasma Pi concentrations were similar under chronic diets, but lower when mice were acutely switched to LPD. Urinary Pi excretion was similar in C57/BL6 and NaPi-IIa(-/-) mice under HPD. During chronic LPD, NaPi-IIa(-/-) mice lost phosphate in urine compensated by higher intestinal Pi absorption. During the acute HPD-to-LPD switch, NaPi-IIa(-/-) mice exhibited a delayed decrease in urinary Pi excretion. PTH was acutely regulated by low dietary Pi intake. FGF23 did not respond to low Pi intake within 8 h whereas the phospho-adaptator protein FRS2α necessary for FGF-receptor cell signaling was downregulated. BBM Pi transport activity and NaPi-IIa but not NaPi-IIc and Pit-2 abundance acutely adapted to diets in C57/BL6 mice. In NaPi-IIa(-/-), Pi transport activity was low and did not adapt. Thus, NaPi-IIa mediates the fast adaptation to Pi intake and is upregulated during the adaptation to low Pi despite persistently high FGF23 levels. The sensitivity to FGF23 may be regulated by adapting FRS2α abundance and phosphorylation.
Asunto(s)
Adaptación Fisiológica , Factores de Crecimiento de Fibroblastos/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas de la Membrana/metabolismo , Fósforo Dietético/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factor-23 de Crecimiento de Fibroblastos , Absorción Intestinal , Túbulos Renales Proximales/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fósforo Dietético/sangre , Fósforo Dietético/orina , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Transport of inorganic phosphate (P(i)) is mediated by proteins belonging to two solute carrier families (SLC20 and SLC34). Members of both families transport P(i) using the electrochemical gradient for Na(+). The role of the SLC34 members as essential players in mammalian P(i) homeostasis is well established, whereas that of SLC20 proteins is less well defined. The SLC34 family comprises the following three isoforms that preferentially cotransport divalent P(i) and are expressed in epithelial tissue: the renal NaPi-IIa and NaPi-IIc are responsible for reabsorbing P(i) in the proximal tubule, whereas NaPi-IIb is more ubiquitously expressed, including the small intestine, where it mediates dietary P(i) absorption. The SLC20 family comprises two members (PiT-1, PiT-2) that preferentially cotransport monovalent P(i) and are expressed in epithelial as well as nonepithelial tissue. The transport kinetics of members of both families have been characterized in detail using heterologous expression in Xenopus oocytes. For the electrogenic NaPi-IIa/b, and PiT-1,-2, conventional electrophysiological techniques together with radiotracer methods have been applied, as well as time-resolved fluorometric measurements that allow new insights into local conformational changes of the protein during the cotransport cycle. For the electroneutral NaPi-IIc, conventional tracer uptake and fluorometry have been used to elucidate its transport properties. The 3-D structures of these proteins remain unresolved and structure-function studies have so far concentrated on defining the topology and identifying sites of functional importance.
Asunto(s)
Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/química , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Animales , Cationes/metabolismo , Túbulos Renales Proximales/metabolismo , Cinética , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa-expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3(KI) mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3(KI) animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3(KI) mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3(KI) mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss.
Asunto(s)
Calcio/orina , Glucógeno Sintasa Quinasa 3/fisiología , Hipofosfatemia Familiar/etiología , Proteínas Inmediatas-Precoces/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Densidad Ósea , Calcitriol/sangre , Túbulos Renales/metabolismo , Ratones , Hormona Paratiroidea/sangre , Fosfatos/metabolismo , FosforilaciónRESUMEN
Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.
Asunto(s)
Densidad Ósea/fisiología , Hipofosfatemia Familiar/etiología , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Transporte Biológico Activo , Densidad Ósea/genética , Calcio/metabolismo , Femenino , Humanos , Hipofosfatemia Familiar/enzimología , Hipofosfatemia Familiar/genética , Técnicas In Vitro , Túbulos Renales/metabolismo , Ratones , Ratones Noqueados , Oocitos/metabolismo , Fosfatos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , XenopusRESUMEN
We have recently shown that the abundance of the renal sodium (Na)/inorganic phosphate (Pi) cotransporter NaPi-IIa is increased in the absence of the GABA(A) receptor-associated protein (GABARAP). Accordingly, GABARAP-deficient mice have a reduced urinary excretion of Pi. However, their circulating levels of Pi do not differ from wild-type animals, suggesting the presence of a compensatory mechanism responsible for keeping serum Pi values constant. Here, we aimed first to identify the molecular basis of this compensation by analyzing the expression of Na/Pi cotransporters known to be expressed in the kidney and intestine. We found that, in the kidney, the upregulation of NaPi-IIa is not accompanied by changes on the expression of either NaPi-IIc or PiT2, the other cotransporters known to participate in renal Pi reabsorption. In contrast, the intestinal expression of NaPi-IIb is downregulated in mutant animals, suggesting that a reduced intestinal absorption of Pi could contribute to maintain a normophosphatemic status despite the increased renal retention. The second goal of this work was to study whether the alterations on the expression of NaPi-IIa induced by chronic dietary Pi are impaired in the absence of GABARAP. Our data indicate that, in response to high Pi diets, GABARAP-deficient mice downregulate the expression of NaPi-IIa to levels comparable to those seen in wild-type animals. However, in response to low Pi diets, the upregulation of NaPi-IIa is greater in the mutant mice. Thus, both the basal expression and the dietary-induced upregulation of NaPi-IIa are increased in the absence of GABARAP.
Asunto(s)
Proteínas del Citoesqueleto/deficiencia , Íleon/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/deficiencia , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo , Sodio/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Densidad Ósea , Proteínas del Citoesqueleto/genética , Fémur/metabolismo , Regulación de la Expresión Génica , Absorción Intestinal , Vértebras Lumbares/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos , Microvellosidades/metabolismo , Fosfatos/sangre , Fosfatos/orina , Fósforo Dietético/metabolismo , ARN Mensajero/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genéticaRESUMEN
Renal phosphate reabsorption across the brush border membrane (BBM) in the proximal tubule is mediated by at least three transporters, NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Parathyroid hormone (PTH) is a potent phosphaturic factor exerting an acute and chronic reduction in proximal tubule phosphate reabsorption. PTH acutely induces NaPi-IIa internalization from the BBM and lysosomal degradation, but its effects on NaPi-IIc and Pit-2 are unknown. In rats adapted to low phosphate diet, acute (30 and 60 min) application of PTH decreased BBM phosphate transport rates both in the absence and the presence of phosphonoformic acid, an inhibitor of SLC34 but not SLC20 transporters. Immunohistochemistry showed NaPi-IIa expression in the S1 to the S3 segment of superficial and juxtamedullary nephrons; NaPi-IIc was only detectable in S1 segments and Pit-2 in S1 and weakly in S2 segments of superficial and juxtamedullary nephrons. PTH reduced NaPi-IIa staining in the BBM with increased intracellular and lysosomal appearance. NaPi-IIa internalization was most prominent in S1 segments of superficial nephrons. We did not detect changes in NaPi-IIc and Pit-2 staining over this time period. Blockade of lysosomal protein degradation with leupeptin revealed NaPi-IIa accumulation in lysosomes, but no lysosomal staining for NaPi-IIc or Pit-2 could be detected. Immunoblotting of BBM confirmed the reduction in NaPi-IIa abundance and the absence of any effect on NaPi-IIc expression. Pit-2 protein abundance was also significantly reduced by PTH. Thus, function and expression of BBM phosphate cotransporters are differentially regulated allowing for fine-tuning of renal phosphate reabsorption.
Asunto(s)
Riñón/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato/metabolismo , Animales , Riñón/ultraestructura , Lisosomas/metabolismo , Masculino , Microvellosidades/metabolismo , Fosfatos/metabolismo , Ratas , Ratas Wistar , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismoRESUMEN
Protein kinases catalyze the phosphorylation of serine/threonine or tyrosine residues, which may directly alter a protein's functional properties. Kinases can also regulate protein functions indirectly, for example, by controlling the composition and/or subcellular localization of members of multiprotein complexes that associate with the regulated protein. In this issue of the JCI, two separate studies by Weinman et al. and Yang et al. examine the second of these two modes of kinase-mediated regulation and demonstrate the effects of kinases on two Na(+)-driven renal cotransporters (see the related articles beginning on pages 3403 and 3412). Their results reveal important implications for phosphate and salt homeostasis, respectively.
Asunto(s)
Transporte Iónico/fisiología , Riñón , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Homeostasis , Riñón/enzimología , Riñón/metabolismo , Fosfatos/metabolismo , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Control of phosphate (P(i)) homeostasis is essential for many biologic functions and inappropriate low levels of P(i) in plasma have been suggested to associate with several pathological states, including renal stone formation and stone recurrence. P(i) homeostasis is achieved mainly by adjusting the renal reabsorption of P(i) to the body's requirements. This task is performed to a major extent by the Na/Pi cotransporter NaPi-IIa that is specifically expressed in the brush border membrane of renal proximal tubules. While the presence of tight junctions in epithelial cells prevents the diffusion and mixing of the apical and basolateral components, the location of a protein within a particular membrane subdomain (i.e., the presence of NaPi-IIa at the tip of the apical microvilli) often requires its association with scaffolding elements which directly or indirectly connect the protein with the underlying cellular cytoskeleton. NaPi-IIa interacts with the four members of the Na(+)/H(+) exchanger regulatory factor family as well as with the GABA(A)-receptor associated protein . Here we will discuss the most relevant findings regarding the role of these proteins on the expression and regulation of the cotransporter, as well as the impact that their absence has in P(i) homeostasis.
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Riñón/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/fisiología , Absorción , Animales , Homeostasis , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Dominios PDZ/fisiología , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
In kidneys of mammals, filtered phosphate ions (Pi) are reabsorbed along the proximal tubules. Transcellular transport of phosphate is initiated by several apically localized sodium-dependent Pi cotransporters (Na/Pi-cotransporters) that belong to the SLC 20 (SLC20A2) and 34 (SLC34A1, SLC34A3) families. Apical abundance of these Na/Pi-cotransporters is adjusted by numerous hormones/phosphatonins and metabolic factors in order to adjust the extent of renal Pi reabsorption according to body needs. Acute hormonal regulation of Pi reabsorption occurs mainly by a change of the abundance of SLC34A1 (NaPi-IIa) via modulation of the interaction of NaPi-IIa with the PDZ-protein NHERF1.
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Riñón/metabolismo , Fosfatos/metabolismo , Absorción , Animales , Dopamina/fisiología , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico , Hormona Paratiroidea/fisiología , Fosfatos/administración & dosificación , Fosfoproteínas/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato/fisiologíaRESUMEN
PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Dominios PDZ , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Animales , Células Cultivadas , Clonación Molecular , Femenino , Humanos , Mamíferos/metabolismo , Ratones , Modelos Biológicos , Oocitos/química , Plásmidos/síntesis química , Unión Proteica , Ratas , Saccharomyces cerevisiae , XenopusRESUMEN
AIM: Several Na+ -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis. METHODS: Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice. Mice were fed 2 weeks standard or low phosphate diets. Stool, urine, plasma and intestinal and renal tissue were collected. Concentration of electrolytes and hormones, expression of mRNAs and proteins and intestinal transport of tracers were analysed. RESULTS: Intestinal mRNA expression of NaPi-IIb and Pit-1 is segment-specific, whereas the abundance of Pit-2 mRNA is more homogeneous. In ileum, NaPi-IIb mRNA expression is restricted to enterocytes, whereas Pit-2 mRNA is found in epithelial and non-epithelial cells. Overall, their mRNA expression is not regulated by dietary phosphate. The absence of Pit-2 from intestinal epithelial cells does not affect systemic phosphate homeostasis under normal dietary conditions. However, in response to dietary phosphate restriction, Pit-2-deficient mice showed exacerbated hypercalciuria and sustained elevation of 1,25(OH)2 vitamin D3 . CONCLUSIONS: In mice, the intestinal Na+ /phosphate cotransporters are not coexpressed in all segments. NaPi-IIb but not Pit-2 mRNA is restricted to epithelial cells. Intestinal epithelial Pit-2 does not contribute significantly to absorption of phosphate under normal dietary conditions. However, it may play a more significant role upon dietary phosphate restriction.