Effects of Npt2 gene ablation and low-phosphate diet on renal Na(+)/phosphate cotransport and cotransporter gene expression.
The renal Na(+)/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na(+)/Pi cotransport, expression of Na(+)/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na(+)/Pi cotransport declines with age in wild-type mice (Npt2(+/+)), but not in mice homozygous for the disrupted Npt2 allele (Npt2(-/-)). At all ages, Na(+)/Pi cotransport in Npt2(-/-) mice is approximately 15% of that in Npt2(+/+) littermates. Only Npt1 mRNA abundance increases with age in Npt2(+/+) mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2(-/-) mice. Pi deprivation significantly increases Na(+)/Pi cotransport, Npt2 protein, and mRNA in Npt2(+/+) mice. In contrast, Pi-deprived Npt2(-/-) mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na(+)/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2(-/-) mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na(+)/Pi cotransport response to Pi deprivation. (+info)
Identification of regulatory sequences and binding proteins in the type II sodium/phosphate cotransporter NPT2 gene responsive to dietary phosphate.
Dietary phosphate (P(i)) is a most important regulator for renal P(i) reabsorption. The type II sodium-dependent phosphate (Na/P(i)) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P(i) reabsorption. The consumption of a low-P(i) diet induces the synthesis of NPT2, whereas a high P(i) diet decreases it. The molecular mechanisms of regulation by dietary P(i) are not yet known. In this report, in weaning mice fed a low-P(i) diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P(i) diet. This increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P(i). The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5'-CACGTG-3'. A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor muE3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the muE3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited the binding to the PRE. The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity. The feeding of a low P(i) diet significantly increased the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P(i). (+info)
NaPO(4) cotransport type III (PiT1) expression in human embryonic kidney cells and regulation by PTH.
The aim of the present study was to characterize the type(s) of NaPO(4) cotransporter expressed in the human renal cell line HEK-293 and its regulation by parathyroid hormone (PTH) in wild-type cells and in cells transfected by the PTH/PTH-related protein (PTHrP) receptor. The results showed that human embryonic kidney HEK-293 cells expressed NaPO(4) cotransporter type III (PiT1) mRNA and protein. In contrast, type I (NPT1) or II (NPT2) cotransporter mRNA were not expressed. Na(+)-dependent phosphate uptake followed a Michaelis-Menten model (apparent maximal transport rate and affinity constant: 23.32 +/- 0.69 nmol PO(4). mg protein(-1). 10 min(-1) and 0.147 +/- 0.014 mM KH(2)PO(4), respectively), was stimulated by phosphate deprivation (maximal increase 24.5 +/- 0.8%, P < 0.001, after 15 h of phosphate deprivation), and was inhibited by increasing pH (3.6 +/- 0.2-fold decrease at pH 8.5, P < 0.0001). It was inhibited in a time- and concentration-dependent fashion by PTH in HEK-293 cells stably transfected by PTH/PTHrP receptors but not in parental HEK-293 cells. Maximal inhibition of Na(+)-dependent phosphate transport was observed at 30 min after the addition of 72 nM PTH-(1-34) (31.5 +/- 2.4% inhibition, P < 0.01). PTH inhibition of phosphate transport was maintained in phosphate-deprived cells and reversed by both GF109203X (10(-6) M) or staurosporine (5.5 nM), two protein kinase C inhibitors. Na(+)-dependent phosphate uptake was also significantly inhibited by phorbol 12-myristate 13-acetate (20.9 +/- 3.9% inhibition, P < 0.001) but not by dibutyril-cAMP (10(-4) M) or forskolin (50 microM). The physiological role played by type III NaPO(4) cotransport expression in the overall renal regulation of phosphate homeostasis remains to be established. (+info)
Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter.
Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5'-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5'-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites. (+info)
Decreased abundance of major Na(+) transporters in kidneys of rats with ischemia-induced acute renal failure.
Ischemia-induced acute renal failure (ARF) is known to be associated with significant impairment of tubular Na reabsorption. We examined whether temporary bilateral renal ischemia (30, 40, or 60 min) and reperfusion (1-5 days) affect the abundance of several renal Na transporters and urinary Na excretion (U(Na)V) in rats. In rats with mild ARF (30 min), immunoblotting revealed that proximal tubule type 3 Na(+)/H(+) exchanger (NHE-3) and type II Na-P(i) cotransporter (NaPi-II) were significantly decreased to 28 +/- 6 and 14 +/- 6% of sham levels, respectively, at day 1. Moreover, Na(+)-K(+)-ATPase levels were also significantly decreased (51 +/- 11%), whereas there was no significant decrease in type 1 bumetanide-sensitive cotransporter (BSC-1) and thiazide-sensitive cotransporter (TSC) levels. Consistent with reduced Na transporter abundance, fractional urinary Na excretion (FE(Na)) was significantly increased in mild ARF (30 min) and U(Na)V was unchanged, despite a marked reduction in glomerular filtration rate. Na transporter levels and renal Na handling were normalized within 5 days. Severe ischemic injury (60 min) resulted in a marked decrease in the abundance of Na(+)-K(+)-ATPase, NHE-3, NaPi-II, BSC-1, and TSC at both days 1 and 5. Consistent with this, FE(Na) was significantly increased at days 1 and 5. Intravenous K-melanocyte-stimulated hormone treatment partially prevented the ischemia-induced downregulation of renal Na transporters and reduced the high FE(Na) to control levels. We conclude that reduced levels of Na transporters along the nephron may play a critical role in the impairment of tubular Na reabsorption, and hence increased Na excretion, in ischemia-induced ARF. (+info)
Altered expression of renal AQPs and Na(+) transporters in rats with lithium-induced NDI.
Lithium (Li) treatment is often associated with nephrogenic diabetes insipidus (NDI). The changes in whole kidney expression of aquaporin-1 (AQP1), -2, and -3 as well as Na-K-ATPase, type 3 Na/H exchanger (NHE3), type 2 Na-Pi cotransporter (NaPi-2), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), and thiazide-sensitive Na-Cl cotransporter (TSC) were examined in rats treated with Li orally for 4 wk: protocol 1, high doses of Li (high Na(+) intake), and protocol 2, low doses of Li (identical food and normal Na(+) intake in Li-treated and control rats). Both protocols resulted in severe polyuria. Semiquantitative immunoblotting revealed that whole kidney abundance of AQP2 was dramatically reduced to 6% (protocol 1) and 27% (protocol 2) of control levels. In contrast, the abundance of AQP1 was not decreased. Immunoelectron microscopy confirmed the dramatic downregulation of AQP2 and AQP3, whereas AQP4 labeling was not reduced. Li-treated rats had a marked increase in urinary Na(+) excretion in both protocols. However, the expression of several major Na(+) transporters in the proximal tubule, loop of Henle, and distal convoluted tubule was unchanged in protocol 2, whereas in protocol 1 significantly increased NHE3 and BSC-1 expression or reduced NaPi-2 expression was associated with chronic Li treatment. In conclusion, severe downregulation of AQP2 and AQP3 appears to be important for the development of Li-induced polyuria. In contrast, the increased or unchanged expression of NHE3, BSC-1, Na-K-ATPase, and TSC indicates that these Na(+) transporters do not participate in the development of Li-induced polyuria. (+info)
Hereditary hypophosphatemic rickets with hypercalciuria is not caused by mutations in the Na/Pi cotransporter NPT2 gene.
Hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a renal phosphate (Pi) wasting disease first described in an extended Bedouin kindred, is characterized by hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D levels, hypercalciuria, rickets, and osteomalacia. Correction of all abnormalities, except for renal Pi wasting, can be achieved by oral Pi supplementation. These findings and the demonstration that mice that are homozygous for the disrupted Na/Pi cotransporter gene Npt2 exhibit many of the biochemical features of HHRH suggested that mutations in the human orthologue NPT2 might be responsible for HHRH. The NPT2 gene in affected individuals from the Bedouin kindred and four small families was screened for mutations to test this hypothesis. No putative disease-causing mutation was found. Two single nucleotide polymorphisms (SNP), a silent substitution in exon 7 and a nucleotide substitution in intron 4, were identified, and neither consistently segregated with HHRH in the Bedouin kindred. Linkage analysis indicated that the two NPT2 intragenic SNP as well as five microsatellite markers in the NPT2 gene region were not linked to HHRH in the Bedouin kindred. Therefore, this is evidence to exclude NPT2 as a candidate gene for HHRH in the families that were studied. (+info)
Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene.
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)