A dibasic motif involved in parathyroid hormone-induced down-regulation of the type IIa NaPi cotransporter.
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Type II NaPi cotransporters are expressed in the apical membrane of P(i)-(re)absorbing epithelia: the type IIa in renal proximal tubule and the type IIb in small intestine. Parathyroid hormone (PTH) leads to a retrieval from the apical membrane of the type IIa NaPi cotransporter. The type IIa cotransporter is also expressed in opossum kidney (OK) cells, and its expression is under the control of PTH. In the present study, we identified the molecular "domains" involved in the PTH-induced retrieval of the type IIa NaPi cotransporter. Wild-type mouse type IIa (mIIa) and type IIb (mIIb) as well as several mIIa-mIIb chimeras and site-directed mutants were fused to the enhanced green fluorescent protein and transfected into OK cells. We found that mIIa but not mIIb was internalized and degraded after incubation with 1-34 (or 3-34) PTH. Using chimeras, we found that the N and C termini were not required in this effect, whereas a "domain" located between residues 216 and 658 seemed to be necessary. This region contains two putative intracellular loops with highly conserved sequences between mIIa and mIIb; in the last intracellular loop, two charged amino acids of type IIa (K(503)R(504)) are replaced by uncharged residues in type IIb (N(520)I(521)). We generated two mutants in which these residues were interchanged: mIIaNI and mIIbKR. Similarly to mIIa, the mIIbKR mutant was endocytosed in response to 1-34 PTH; in contrast, mIIaNI behaved as mIIb and was not internalized. In conclusion, a dibasic amino acid motif (K(503)R(504)) located in the last intracellular loop of the type IIa NaPi cotransporter is essential for its PTH-induced retrieval. (+info)
Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene.
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BACKGROUND: At present the genetic defect for autosomal recessive and autosomal dominant hypophosphataemic rickets with hypercalciuria (HHRH) is unknown. Type II sodium/phosphate cotransporter (NPT2) gene is a serious candidate for being the causative gene in either or both autosomal recessive and autosomal dominant HHRH. In the present study we tested this hypothesis in one autosomal recessive family. METHODS: The gene structure of human NPT2 is known. We tested the complete open reading frame in the affected siblings by polymerase chain reaction in combination with automatic DNA sequencing for the presence of mutations. RESULTS: We did not observe disease-causing mutations in the NPT2 gene of the affected siblings. A T855C polymorphism resulting in a histidine to arginine transition was present in the open reading frame of NPT2. The polymorphism was present in both affected as well as unaffected family members. CONCLUSION: The hypothesis that a defect in the NPT2 gene could be an underlying cause for autosomal recessive HHRH could not be sustained in our study. (+info)
Amino acids involved in sodium interaction of murine type II Na(+)-P(i) cotransporters expressed in Xenopus oocytes.
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Type IIa and IIb Na+-Pi cotransporters are highly conserved proteins expressed in brush border membranes of proximal tubules and small intestine, respectively. The kinetics of IIa and IIb differ significantly: type IIb is saturated at lower concentrations of Na+ and Pi. To define the domain responsible for the difference in Na+ affinity we constructed several mouse IIa-IIb chimeras as well as site-directed mutagenized cotransporters. Pi uptake activity was determined after injection of cRNAs into Xenopus laevis oocytes. From the chimera experiments we concluded that the domain containing part of the second intracellular loop, the fifth transmembrane domain (TD) and part of the third extracellular loop determines the specific Na+ activation properties for both types of cotransporter. Within this domain only a few residues located in the fifth TD are not conserved between type IIa and IIb. Site-directed mutagenesis on non-conserved residues was performed. Substitution of F402 of IIa by the corresponding L418 from IIb yielded a cotransporter that behaved like the IIb. On the other hand, substitution of the specific L418 of IIb by the corresponding F402 of IIa produced a cotransporter with a Na+ activation similar to IIa. (Single letter amino acid nomenclature is used throughout the paper.) These data suggest that the specific Na+ activation properties exhibited by type IIa and type IIb Na+-Pi cotransporters are at least in part due to the presence of a specific amino acid (F402 in IIa, and L418 in IIb) within the fifth TD of the protein. (+info)
Gentamicin causes endocytosis of Na/Pi cotransporter protein (NaPi-2).
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BACKGROUND: Renal toxicity is a major side-effect of aminoglycoside antibiotics and is characterized by an early impairment in proximal tubular function. In a previous study, we have shown that gentamicin administration to the rat causes an early impairment in sodium gradient-dependent phosphate (Na/Pi) cotransport activity. The purpose of our current study was to determine the molecular mechanisms of the impairment in Na/Pi cotransport activity, specifically the role of the proximal tubular type II Na/Pi cotransporter. METHODS: Rats were treated for one, two, and three days with two daily injections of 30 mg/kg body weight gentamicin or the vehicle. RESULTS: Gentamicin caused a progressive decrease in superficial cortical apical brush-border membrane (SC-BBM) Na/Pi cotransporter activity (856 +/- 93 in control vs. 545 +/- 87 pmol/mg BBM protein in 3-day gentamicin, P < 0.01). Western blot analysis showed a parallel and progressive decrease in SC-BBM Na/Pi cotransporter protein abundance, a 50% decrease after one day of treatment, a 63% decrease after two days of treatment, and an 83% decrease after three days treatment with gentamicin. In contrast, gentamicin treatment had no effect on Na/Pi cotransport activity or Na/Pi cotransporter protein abundance in BBM isolated from the juxtamedullary cortex (JMC-BBM). Immunofluorescence microscopy showed a major decrease in the expression of Na/Pi cotransporter protein in the apical membrane of the proximal convoluted tubule, with progressive intracellular accumulation of Na/Pi protein. Colocalization studies showed that in gentamicin-treated rats, Na/Pi protein was colocalized in the early endosomes and especially in the lysosomes. Northern blot analysis of cortical RNA interestingly showed no reduction in Na/Pi cotransporter mRNA abundance even after three days of gentamicin treatment. CONCLUSION: We conclude that gentamicin inhibits Na/Pi cotransport activity by causing a decrease in the expression of the type II Na/Pi cotransport protein at the level of the proximal tubular apical BBM and that inhibition of Na/Pi cotransport activity is most likely mediated by post-transcriptional mechanisms. (+info)
Transcriptional regulation of the NPT2 gene by dietary phosphate.
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Dietary phosphate (Pi) is an important regulator for renal Pi reabsorption. The type II sodium-dependent phosphate (Na/Pi) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal Pi reabsorption. The yeast one-hybrid system was used to clone a transcription factor that binds to a specific sequence (Pi response element) in the promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor mu E3 (TFE3) were isolated. TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary Pi. (+info)
Glycosphingolipids modulate renal phosphate transport in potassium deficiency.
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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. (+info)
Vitamin D reduces renal NaPi-2 in PTH-infused rats: complexity of vitamin D action on renal P(i) handling.
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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. (+info)
Evidence for a PTH-independent humoral mechanism in post-transplant hypophosphatemia and phosphaturia.
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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. (+info)