Npt2a Gene Research Articles

Parathyroid Hormone (PTH) decreases mRNA stability of the Type IIa Sodium‐Phosphate Cotransporter (NpT2a)

In primary hyperparathyroidism, chronically elevated levels of PTH produce low serum phosphorus through inhibition of proximal tubule expression of sodium‐phosphate cotransporters. We have previously shown that chronic PTH stimulation of proximal tubule cells is associated with decreased expression of NpT2a mRNA through destabilization, which is dependent on transcription. BLAST evaluation of the NpT2a promoter identified a CREB‐binding region. We hypothesize that PTH decreases NpT2a mRNA stability through activation of the PKA pathway. Opossum kidney (OK) cells were transfected with luciferase reporter gene constructs containing either full‐length or serially truncated NpT2a promoter regions. Luciferase reporter assays show no effect of PTH on the promoter function of the NpT2a gene. Direct activation of PKA with 8Br‐cAMP and PKC with PMA for 2h produced a decrease in NpT2a mRNA of 35.6 ± 12.2% and 28.9 ± 4.3%, respectively. We conclude that PTH decreases NpT2a mRNA expression through destabilization of NpT2a mRNA, and that both PKA and PKC pathways may contribute to this effect. Support for this project is provided by VA to EDL.

Hypercholesterolemia and effects of high cholesterol diet in type IIa sodium-dependent phosphate co-transporter (Npt2a) deficient mice

The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol.

Regulation of renal sodium-dependent phosphate co-transporter genes (Npt2a and Npt2c) by all-trans-retinoic acid and its receptors

The type II sodium-dependent phosphate co-transporters Npt2a and Npt2c play critical roles in the reabsorption of Pi by renal proximal tubular cells. The vitamin A metabolite ATRA (all-trans-retinoic acid) is important for development, cell proliferation and differentiation, and bone formation. It has been reported that ATRA increases the rate of Pi transport in renal proximal tubular cells. However, the molecular mechanism is still unknown. In the present study, we observed the effects of a VAD (vitamin A-deficient) diet on Pi homoeostasis and the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush-border membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of human Npt2a and Npt2c genes was increased markedly by ATRA and a RAR (retinoic acid receptor)-specific analogue TTNPB {4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} in renal proximal tubular cells overexpressing RARs and RXRs (retinoid X receptors). Furthermore, we identified RAREs (retinoic acid-response elements) in both gene promoters. Interestingly, the half-site sequences (5'-GGTTCA-3': -563 to -558) of 2c-RARE1 overlapped the vitamin D-responsive element in the human Npt2c gene and were functionally important motifs for transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 (1alpha,25-dihydroxyvitamin D3), in both independent or additive actions. In summary, we conclude that VAD induces hyperphosphaturia through the down-regulation of Npt2a and Npt2c gene expression in the kidney.

Transgenic mice overexpressing secreted frizzled-related proteins (sFRP)4 under the control of serum amyloid P promoter exhibit low bone mass but did not result in disturbed phosphate homeostasis

Secreted frizzled-related protein-4 (sFRP4) is a member of secreted modulators of Wnt signaling pathways and has been recognized to play important role in the pathogenesis of oncogenic osteomalacia as a potential phosphatonin. To investigate the role of sFRP4 in bone biology and phosphorus homeostasis in postnatal life, we generated transgenic mice that overexpress sFRP4 under the control of the serum amyloid P promoter (SAP-sFRP4), which drives transgene expression postnatally. Serum phosphorus level and urinary phosphorus excretion were slightly lower and higher, respectively, in SAP-sFRP4 compared to wild-type (WT) littermate, but the difference did not reach statistical significance. However, renal Na ±/Pi co-transporter (Npt) 2a and 1α-hydroxylase gene expression were up-regulated in SAP-sFRP4 mice. In addition, the level of serum 1,25-dihydroxyvitamin D 3 was higher in SAP-sFRP4 mice. At 5 weeks of age, bone mineral density (BMD) in SAP-sFRP4 was similar to that in WT. However, with advancing age, SAP-sFRP4 mice gained less BMD so that areal BMD of SAP-sFRP4 mice was significantly lower compared to WT at 15 weeks of age. Histomorphometric analysis of proximal tibia showed that trabecular bone volume (BV/TV) and thickness (Tb.Th) were significantly lower in SAP-sFRP4 mice. There was no evidence of osteomalacia in histological analysis. Our data do not support the role of sFRP4 per se as a phosphatonin but suggest that sFRP4 negatively regulates bone formation without disrupting phosphorus homeostasis.

Thyroid hormones regulate phosphate homoeostasis through transcriptional control of the renal type IIa sodium-dependent phosphate co-transporter (Npt2a) gene

The type IIa renal sodium-dependent phosphate (Na/Pi) co-transporter Npt2a is implicated in the control of serum phosphate levels. It has been demonstrated previously that renal Npt2a protein and its mRNA expression are both up-regulated by the thyroid hormone T3 (3,3',5-tri-iodothyronine) in rats. However, it has never been established whether the induction was mediated by a direct effect of thyroid hormones on the Npt2a promoter. To address the role of Npt2a in T3-dependent regulation of phosphate homoeostasis and to identify the molecular mechanisms by which thyroid hormones modulate Npt2a gene expression, mice were rendered pharmacologically hypo- and hyper-thyroid. Hypothyroid mice showed low levels of serum phosphate and a marked decrease in renal Npt2a protein abundance. Importantly, we also showed that Npt2a-deficient mice had impaired serum phosphate responsiveness to T3 compared with wild-type mice. Promoter analysis with a luciferase assay revealed that the transcriptional activity of a reporter gene containing the Npt2a promoter and intron 1 was dependent upon TRs (thyroid hormone receptors) and specifically increased by T3 in renal cells. Deletion analysis and EMSAs (electrophoretic mobility-shift assays) determined that there were unique TREs (thyroid-hormone-responsive elements) within intron 1 of the Npt2a gene. These results suggest that Npt2a plays a critical role as a T3-target gene, to control phosphate homoeostasis, and that T3 transcriptionally activates the Npt2a gene via TRs in a renal cell-specific manner.

NPT2a gene variation in calcium nephrolithiasis with renal phosphate leak

A decrease in renal phosphate reabsorption with mild hypophosphatemia (phosphate leak) is found in some hypercalciuric stone-formers. The NPT2a gene encodes a sodium-phosphate cotransporter, located in the proximal tubule, responsible for reclaiming most of the filtered phosphate load in a rate-limiting manner. To determine whether genetic variation of the NPT2a gene is associated with phosphate leak and hypercalciuria in a cohort of 98 pedigrees with multiple hypercalciuric stone-formers, we sequenced the entire cDNA coding region of 28 probands, whose tubular reabsorption of phosphate normalized for the glomerular filtration rate (TmP/GFR) was 0.7 mmol/l or lower. We performed genotype/phenotype correlations for each genetic variant in the entire cohort and expressed NPT2a variant RNAs in Xenopus laevis oocytes to test for cotransporter functionality. We identified several variants in the coding region including an in-frame 21 bp deletion truncating the N-terminal cytoplasmic tail of the protein (91del7), as well as other single-nucleotide polymorphisms that were non-synonymous (A133V and H568Y) or synonymous. Levels of TmP/GFR and urine calcium excretion were similar in heterozygote carriers of NPT2a variants compared to the wild-type (wt) homozygotes. The transport activity of the H568Y mutants was identical to the wt, whereas the N-terminal-truncated version and the 91del7 and A133V mutants presented minor kinetic changes and a reduction in the expression level. Although genetic variants of NPT2a are not rare, they do not seem to be associated with clinically significant renal phosphate or calcium handling anomalies in a large cohort of hypercalciuric stone-forming pedigrees.

Alternative promoters and renal cell-specific regulation of the mouse type IIa sodium-dependent phosphate cotransporter gene

The type IIa sodium-dependent phosphate cotransporter (NPT2a) expressed in renal proximal tubules represents an important determinant in maintaining inorganic phosphate (Pi) homeostasis. In the present study, we identified two variant transcripts of the mouse NPT2a gene, Npt2a-v1 and Npt2a-v2, characterized by the presence of alternative first exons (either exon 1A or exon 1B). The chromosomal structure analysis revealed that the Npt2a gene comprises of two promoters (promoters 1 and 2) and 14 exons, and spans approximately 17 kb. Quantitative PCR analysis showed that renal mRNA levels of both the variants markedly decreased in X-linked vitamin D-resistant hypophosphatemic rickets (Hyp) mice compared to normal littermates. Interestingly, transcriptional activity of a reporter gene, containing Npt2a promoters 1 and 2, was renal cell—specifically increased by 1α, 25(OH) 2D 3 and its analogs. The deletion analysis revealed that the CAAT box in the Npt2a promoter 2 is important for the 1α, 25(OH) 2D 3-dependent renal cell-specific activation of the reporter gene. These data suggested that two alternative promoters control the renal expression of Npt2a gene and both Npt2a variant transcripts are down regulated in Hyp mice.

REGULATION OF PHOSPHORUS HOMEOSTASIS BY THE TYPE IIA NA/PHOSPHATE COTRANSPORTER

The type IIa Na/phosphate (Pi) cotransporter (Npt2a) is expressed in the brush border membrane (BBM) of renal proximal tubular cells where the bulk of filtered Pi is reabsorbed. Disruption of the Npt2a gene in mice elicits hypophosphatemia, renal Pi wasting, and an 80% decrease in renal BBM Na/Pi cotransport, and led to the demonstration that Npt2a is the target for hormonal and dietary regulation of renal Pi reabsorption. Regulation is achieved by changes in BBM abundance of Npt2a protein and requires the interaction of Npt2a with various scaffolding and regulatory proteins. Molecular studies in patients with renal Pi wasting resulted in the identification of novel regulators of Pi homeostasis: fibroblast growth factor-23 (FGF-23) and a phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). In mouse models, increased FGF-23 production or loss of Phex function causes hypophosphatemia and decreased renal Pi reabsorption, secondary to decreased BBM Npt2a protein abundance. Thus, Npt2a plays a major role in the maintenance of Pi homeostasis in both health and disease.

Sodium-phosphate cotransporters, nephrolithiasis and bone demineralization.

We discuss how recent findings obtained in disorders of phosphate metabolism in humans and in animal models have provided insights into the pathogenesis of renal stone formation and bone demineralization. Mice that are null for the sodium-phosphate cotransporter (NPT)2a gene (NPT2a(-/-) mice) exhibit hypophosphataemia, increased urinary phosphate excretion, hypercalciuria and nephrolithiasis, but no bone demineralization. Mice null for the sodium-hydrogen exchanger regulatory factor (NHERF)1 (NHERF1(-/-) mice) also exhibit hypophosphataemia and increased renal phosphate excretion with decreased renal NPT2a expression, but they present with a severe sex-dependent bone demineralization. Heterozygous loss-of-function mutations in the NPT2a gene in humans induce hypophosphataemia, increased urinary phosphate excretion, hypercalciuria, nephrolithiasis in males (to date) and bone demineralization of variable severity in both sexes. Patients and experimental animals with increased circulating levels of fibroblast growth factor 23 present with hypophosphataemia, increased urinary phosphate excretion, inappropriate calcitriol synthesis and rickets/osteomalacia, but no nephrolithiasis except when treated. Low-phosphate diet in spontaneously hypercalciuric rats and disruption of the 1-alpha-hydroxylase gene in NPT2a mice prevent renal stone formation. Increased urinary phosphate excretion is a risk factor for renal calcium stone formation when it is associated with hypercalciuria. As yet undefined interplay between NPT2a, NHERF1 and possibly other cotransporters or associated proteins in bone cells may account for the diversity of bone phenotypes observed in disorders of phosphate metabolism with impaired renal phosphate reabsorption. The pathogenesis of both renal stone and bone demineralization appear to be affected by species, sex and mutation type, among other factors.

Differential effects of Npt2a gene ablation and X-linked Hyp mutation on renal expression of Npt2c.

The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (Pi) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-Pi cotransport. In mice homozygous for the disrupted Npt2a gene (Npt2-/-), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2+/+ littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to beta-actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. Pi deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2+/+ mice but failed to elicit a further increase in Npt2c protein in Npt2-/- mice. Pi restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2-/- mice and Hyp mice and that the Npt2c response to low-Pi challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2-/- mice and suggest that Npt2c likely accounts for residual BBM Na-Pi cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression.

Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter.

Epidemiologic studies suggest that genetic factors confer a predisposition to the formation of renal calcium stones or bone demineralization. Low serum phosphate concentrations due to a decrease in renal phosphate reabsorption have been reported in some patients with these conditions, suggesting that genetic factors leading to a decrease in renal phosphate reabsorption may contribute to them. We hypothesized that mutations in the gene coding for the main renal sodium-phosphate cotransporter (NPT2a) may be present in patients with these disorders. We studied 20 patients with urolithiasis or bone demineralization and persistent idiopathic hypophosphatemia associated with a decrease in maximal renal phosphate reabsorption. The coding region of the gene for NPT2a was sequenced in all patients. The functional consequences of the mutations identified were analyzed by expressing the mutated RNA in Xenopus laevis oocytes. Two patients, one with recurrent urolithiasis and one with bone demineralization, were heterozygous for two distinct mutations. One mutation resulted in the substitution of phenylalanine for alanine at position 48, and the other in a substitution of methionine for valine at position 147. Phosphate-induced current and sodium-dependent phosphate uptake were impaired in oocytes expressing the mutant NPT2a. Coinjection of oocytes with wild-type and mutant RNA indicated that the mutant protein had altered function. Heterozygous mutations in the NPT2a gene may be responsible for hypophosphatemia and urinary phosphate loss in persons with urolithiasis or bone demineralization.