Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (P(i)) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal P(i) diet (NPD, 0.6% P(i)) or low P(i) diet (0.07% P(i)) for 2 days, in the presence and absence of parathyroid hormone (PTH). P(i) reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater P(i) uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary P(i) restriction in juvenile rats resulted in a significant increase in P(i) reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that P(i) reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary P(i) restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular P(i) reabsorption.     

A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism

Background

The sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule.

Methods

We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production.

Results

We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a.

Conclusions

Our results indicate that the PDZ1 domain is critical for NHERF1- NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.     

ASARM peptides: PHEX-dependent and -independent regulation of serum phosphate

Increased acidic serine aspartate-rich MEPE-associated motif (ASARM) peptides cause mineralization defects in X-linked hypophosphatemic rickets mice (HYP) and "directly" inhibit renal phosphate uptake in vitro. However, ASARM peptides also bind to phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and are a physiological substrate for this bone-expressed, phosphate-regulating enzyme. We therefore tested the hypothesis that circulating ASARM peptides also "indirectly" contribute to a bone-renal PHEX-dependent hypophosphatemia in normal mice. Male mice (n = 5; 12 wk) were fed for 8 wk with a normal phosphorus and vitamin D(3) diet (1% P(i) diet) or a reduced phosphorus and vitamin D(3) diet (0.1% P(i) diet). For the final 4 wk, transplantation of mini-osmotic pumps supplied a continuous infusion of either ASARM peptide (5 mg·day(-1)·kg(-1)) or vehicle. HYP, autosomal recessive hypophosphatemic rickets (ARHR), and normal mice (no pumps or ASARM infusion; 0.4% P(i) diet) were used in a separate experiment designed to measure and compare circulating ASARM peptides in disease and health. ASARM treatment decreased serum phosphate concentration and renal phosphate cotransporter (NPT2A) mRNA with the 1% P(i) diet. This was accompanied by a twofold increase in serum ASARM and 1,25-dihydroxy vitamin D(3) [1,25 (OH)(2)D(3)] levels without changes in parathyroid hormone. For both diets, ASARM-treated mice showed significant increases in serum fibroblast growth factor 23 (FGF23; +50%) and reduced serum osteocalcin (-30%) and osteopontin (-25%). Circulating ASARM peptides showed a significant inverse correlation with serum P(i) and a significant positive correlation with fractional excretion of phosphate. We conclude that constitutive overexpression of ASARM peptides plays a "component" PHEX-independent part in the HYP and ARHR hypophosphatemia. In contrast, with wild-type mice, ASARM peptides likely play a bone PHEX-dependent role in renal phosphate regulation and FGF23 expression. They may also coordinate FGF23 expression by competitively modulating PHEX/DMP1 interactions and thus bone-renal mineral regulation.     

Inorganic phosphate homeostasis and the role of dietary phosphorus

Inorganic phosphate (Pi) is required for cellular function and skeletal mineralization. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption. The crucial regulated step in Pi homeostasis is the transport of Pi across the renal proximal tubule. Type II sodium-dependent phosphate (Na/Pi) cotransporter (NPT2) is the major molecule in the renal proximal tubule and is regulated by Pi, parathyroid hormone and by 1,25-dihydroxyvitamin D. Recent studies of inherited and acquired hypophosphatemia [X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced rickets/osteomalacia (TIO)], which exhibit similar biochemical and clinical features, have led to the identification of novel genes, PHEX and FGF23, that play a role in the regulation of Pi homeostasis. The PHEX gene, which is mutated in XLH, encodes an endopeptidase, predominantly expressed in bone and teeth, but not in kidney. FGF-23 may be a substrate of this endopeptidase and may therefore accumulate in patients with XLH. In the case of ADHR mutations in the furin cleavage site, which prevent the processing of FGF-23 into fragments, lead to the accumulation of a "stable" circulating form of the peptide which also inhibits renal Pi reabsorption. In the case of TIO, ectopic overproduction of FGF-23 overwhelms its processing and degradation by PHEX, leading to the accumulation of FGF-23 in the circulation and inhibition of renal Pi reabsorption. Mice homozygous for severely hypomorphic alleles of the Klotho gene exhibit a syndrome resembling human aging, including atherosclerosis, osteoporosis, emphysema, and infertility. The KLOTHO locus is associated with human survival, defined as postnatal life expectancy, and longevity, defined as life expectancy after 75. In considering the relationship of klotho expression to the dietary Pi level, the klotho protein seemed to be negatively controlled by dietary Pi.     

The regulation and function of phosphate in the human body

Inorganic phosphate (Pi) is required for cellular function and skeletal mineralization. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption. Pi is abundant in the diet, and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. The crucial regulated step in Pi homeostasis is the transport of Pi across the renal proximal tubule. Type II sodium-dependent phosphate (Na/Pi) cotransporter (NPT2) is the major molecule in the renal proximal tubule and is regulated by hormones and nonhormonal factors. Recent studies of inherited and acquired hypophosphatemia which exhibit similar biochemical and clinical features, have led to the identification of novel genes, phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and fibroblast growth factor-23 (FGF-23), that play a role in the regulation of Pi homeostasis. The PHEX gene encodes an endopeptidase, predominantly expressed in bone and teeth but not in kidney. FGF-23 may be a substrate of this endopeptidase and inhibit renal Pi reabsorption. In a survey in the United States and in Japan, the amount of phosphorus from food is gradually increasing. It is thought that excess amounts of phosphorus intake for long periods are a strong factor in bone impairment and ageing. The restriction of phosphorus intake seems to be important under low calcium intake to keep QOL on high level.     

Acute regulation of parathyroid hormone by dietary phosphate

Secondary hyperparathyroidism in chronic renal failure is stimulated by dietary phosphate (P(i)) loading and ameliorated by dietary P(i) restriction. We investigated the rapidity of the response of serum parathyroid hormone (PTH) to changes in dietary P(i). When uremic rats adapted to a high P(i) diet (HPD) were fed a single meal of low P(i) diet (LPD), plasma PTH fell 80% within 2 h; plasma P(i) fell 1 mg/dl with no change in plasma ionized Ca (ICa). When uremic rats on the HPD were gavaged with LPD, PTH fell 60% within 15 min; plasma P(i) fell by 3.0 mg/dl with no change in total plasma Ca. However, HPD gavage increased PTH by 80% within 15 min with no change in plasma P or Ca, suggesting that the response may be independent of altered plasma P(i). Duodenal infusion of sodium P(i) increased PTH twofold within 10 min, with no change in ICa but an increase in plasma P(i), whereas duodenal infusion of NaCl had no effect on any of these parameters. Intravenous infusion of sodium phosphate also increased PTH within 10 min with no change in plasma ICa; intravenous NaCl had no effect. Additionally, duodenal infusion of phosphonoformate, a nonabsorbable phosphate analog, increased PTH fourfold within 5 min, but did not change plasma P or ICa. These findings indicate that oral P(i) increases PTH release in vivo more rapidly than previously reported; this response may be from both plasma phosphate and an additional signal arising from the gastrointestinal tract.     

In vivo fractional P(i) absorption and NaPi-II mRNA expression in rainbow trout are upregulated by dietary P restriction

Mammalian type II sodium-phosphate cotransporter (NaPi-II) and inorganic phosphate uptake stimulator (PiUS) genes are upregulated by dietary phosphorus (P) restriction to increase intestinal and renal P transport, but little is known about NaPi-II and PiUS regulation in other vertebrates. We studied the 1). the tissue distribution and dietary regulation of NaPi-II, PiUS, and sodium-glucose cotransporter (SGLT1) mRNA and NaPi-II protein in juvenile rainbow trout (Oncorhynchus mykiss) and 2). effects of dietary P on intestinal Pi absorption in vivo. NaPi-II, PiUS, and SGLT1 mRNA were found in the proximal and distal intestine, pyloric ceca, and kidney. PiUS mRNA was also found in the heart, gill, blood, stomach, liver, skin, and muscle. Tissue distribution of NaPi-II protein correlated with that of NaPi-II mRNA except in gill ionocytes where NaPi-II antibodies recognized related epitopes. Chronic consumption of a low-P diet increased NaPi-II and PiUS but not SGLT1 mRNA abundance in the intestine and kidney. Unlike mammals, there was no detectable shift in tissue or cellular localization of NaPi-II protein in response to dietary P restriction. Regulation of NaPi and PiUS mRNA expression was observed only in fish grown under optimal aqueous oxygen concentrations. In vivo fractional absorption of Pi by the intestine decreased in fish fed high-P diets. Decreases in absorption were less pronounced in fish previously fed low-P diets, suggesting that diet history modulates acute regulation of P absorption. Regulation of dietary Pi absorption in vivo may involve a specific change in intestinal NaPi-II and PiUS gene expression.     

Hormonal regulation of phosphate homeostasis in goats during transition to rumination

Regulatory processes in phosphorus (P) homeostasis in small ruminants are quite different compared to monogastric animals. Adaptive responses of modulating hormones [parathyroid hormone (PTH) and calcitriol] to feeding variable amounts of P are lacking. Therefore, the aim of this study was to examine the influence of high dietary P intake (control diet: 4 g kg(-1) dry matter; high-P diet: 8 g kg(-1) dry matter) on the expression levels of PTH receptor (PTHR), vitamin D receptor (VDR) and Na+-dependent Pi transporters (NaPi II) in kidney and jejunum of goats starting rumination. After 3 months of feeding, plasma phosphate (Pi) and PTH concentrations were increased in the high-P diet group, whereas calcium and calcitriol were not changed. The intestinal Na+-dependent Pi transport capacity was not influenced by a high-P diet and the expression of jejunal VDR, PTHR and NaPi IIb was not modified. Interestingly, renal Na+-dependent Pi transport capacity was significantly reduced and concomitantly the expression of PTHR and NaPi IIa was decreased. In conclusion, the adaptive response of renal Pi reabsorption in goats, which were in transition from non-ruminant to ruminant stage was comparable to that of monogastric animals. In contrast, the modulation of the intestinal Pi absorption was like in adult ruminants.     

Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.     

Combining suppressive subtractive hybridization and cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney

Phosphorus (P)-responsive genes and how they regulate renal adaptation to phosphorous-deficient diets in animals, including fish, are not well understood. RNA abundance profiling using cDNA microarrays is an efficient approach to study nutrient–gene interactions and identify these dietary P-responsive genes. To test the hypothesis that dietary P-responsive genes are differentially expressed in fish fed varying P levels, rainbow trout were fed a practical high-P diet (R20: 0.96% P) or a low-P diet (R0: 0.38% P) for 7 weeks. The differentially-expressed genes between dietary groups were identified and compared from the kidney by combining suppressive subtractive hybridization (SSH) with cDNA microarray analysis. A number of genes were confirmed by real-time PCR, and correlated with plasma and bone P concentrations. Approximately 54 genes were identified as potential dietary P-responsive after 7 weeks on a diet deficient in P according to cDNA microarray analysis. Of 18 selected genes, 13 genes were confirmed to be P-responsive at 7 weeks by real-time PCR analysis, including: iNOS, cytochrome b, cytochrome c oxidase subunit II , α-globin I, β-globin, ATP synthase, hyperosmotic protein 21, COL1A3, Nkef, NDPK, glucose phosphate isomerase 1, Na+/H+ exchange protein and GDP dissociation inhibitor 2. Many of these dietary P-responsive genes responded in a moderate way (R0/R20 ratio: < 2–3 or > 0.5) and in a transient manner to dietary P limitation. In summary, renal adaptation to dietary P deficiency in trout involves changes in the expression of several genes, suggesting a profile of metabolic stress, since many of these differentially-expressed candidates are associated with the cellular adaptative responses.     

Role of PDZK1 protein in apical membrane expression of renal sodium-coupled phosphate transporters

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.     

Sodium-dependent phosphate co-transporters

The renal proximal tubular reabsorption of inorganic phosphate (Pi) mediated by sodium-dependent phosphate (Na+/Pi) co-transporters plays a critical role in the maintenance of Pi homeostasis. Two nonhomologous Na+/Pi co-transporters (type I and type II) have been identified in the renal cortex of various species. The role of the type I co-transporter in Pi regulation remains to be clarified. Type II co-transporters play a major role in the regulation of renal Pi reabsorption by dietary Pi and parathyroid hormone, which regulate the rapid endocytosis/exocytosis of the transporters. Type III Na+/Pi co-transporters, which are expressed in a wide variety of tissues and are regulated by changes in the Pi concentration, have recently been described. The presence of a novel Pi-regulating hormone called 'phosphatonin' has been postulated in studies of the mechanisms of X-linked hypophosphatemic rickets and oncogenic osteomalacia. The regulation of phosphatonin and Na+/Pi co-transporters may provide novel pharmacological approaches to the treatment of these diseases.