In the kidney, filtered inorganic phosphate ions are reabsorbed along the proximal tubules. This transepithelial process involves sodium-dependent phosphate transporters that are localized at the apical (brush border) membrane. Currently, three Na/Pi-cotransporters that belong to the SLC 20 (PiT-2) and SLC 34 (NaPi-lla and NaPi-llc) families have been assigned to proximal tubular phosphate reabsorption, whereby SLC34 proteins play the major role. The primary functional difference between SLC34 and SLC20 proteins is that the former preferentially transport divalent P i whereas the latter prefer monovalent Pi.
Renal excretion of phosphate is controlled by the number of Na/Pi-cotransporters residing in the apical membrane. The abundance of Na/Pi-cotransporters is controlled by a multidue of hormones and metabolic factors. Genetic diseases associated with disturbed phosphate homeostasis affect either Na/Pi-cotransporters directly or the stability and production of regulatory factors.
proximal tubule, phosphate, NaPi-cotransport, NaPi-lla, regulation, NHERF1
The Importance of Phosphorus in Biological Systems
Phosphorus is the sixth most abundant element in the body and is essential for energy-consuming metabolic processes of cells. Approximately 85% of phosphate in the body is present in bones, 14% in cells from soft tissues, and 1% in extracellular fluids. Phosphorus plays an important role in a number of biological processes, and is an exceptionally important component of hy droxyapatite, the major component of bone mineral. In addition, phosphorus is present in nucleic acids, bioactive signaling proteins, phosphorylated enzymes, and cell membranes. A prolonged deficiency of phosphorus and inorganic phosphate results in serious biological problems, including bone mineralization resulting in osteomalacia or rickets, abnormal erythrocyte, leukocyte and platelet function, impaired cell membrane integrity that can result in rhabdomyolysis, and impaired cardiac function. Therefore, the maintenance of appropriate phosphorus homeostasis is critical for the well-being of the organism.
The Regulation of Phosphate Balance
Phosphorus exists in plasma almost entirely as inorganic phosphate. The adult animal or human maintains phosphate balance through a series of complex hormonally and locally regulated metabolic adjustments. In states of neutral phosphate balance, net accretion equals net output. The major organs involved in the absorption, excretion, and reabsorption of phosphate are the intestine and the kidney ( Fig. 69.1 ). Furthermore, the movement of phosphorus between the extracellular fluid and bone and soft tissue also plays an important role in the maintenance of normal serum phosphate concentrations. A normal diet adequate in phosphorus normally contains ≈1500 mg of phosphorus. Approximately 1100 mg of ingested dietary phosphate is absorbed in the proximal intestine, predominantly in the jejunum. About 200 mg of phosphorus is secreted into the intestine via pancreatic and intestinal secretions, giving a net phosphorus absorption of approximately 900 mg/24 hours. Phosphorus that is not absorbed in the intestine or is secreted into the intestinal lumen eventually appears in the feces. Absorbed phosphorus enters the extracellular fluid pool and moves in and out of bone (and to a smaller extent in and out of soft tissues) as needed (≈200 mg). Approximately 900 mg of phosphorus (equivalent to the amount absorbed in the intestine) is excreted in thse urine.
A number of hormones such as parathyroid hormone (PTH) and 1α,25(OH) 2 D 3 are involved in the control of phosphorus metabolism. Concentrations of these hormones are regulated by phosphorus in a manner that is conducive to the maintenance of normal phosphate. Other peptide factors and hormones, such as growth hormone and insulin-like growth factor 1, alter phosphorus balance, although their circulating concentrations are not directly controlled by ambient phosphorus concentrations. The “phosphatonins,” fibroblast growth factor 23 (FGF-23), and secreted frizzled related protein-4 (sFRP-4) that induce a state of negative phosphate balance directly by inhibiting renal phosphate reabsorption in the proximal tubule and indirectly by inhibiting the synthesis of 1α,25(OH) 2 D 3 and reducing the intestinal absorption of phosphorus, also play a key role in the regulation of phosphate balance. Two factors, fibroblast growth factor 7 (FGF-7) and matrix extracellular phosphoglycoprotein (MEPE), have been shown to inhibit phosphate transport in renal epithelial cells in culture and, in the case of matrix extracellular phosphorus glycoprotein, to induce phosphaturia in mice. FGF7 and MEPE, however, have not been demonstrated to prevent compensatory increases in serum 1α,25(OH) 2 D 3 concentrations seen in hypophosphatemic states or to directly inhibit 25-hydroxyvitamin D 1α-hydroxylase activity.
Parathyroid hormone, by virtue of its phosphaturic effect in the kidney, decreases overall phosphate retention, whereas 1α,25(OH) 2 D 3 increases phosphate retention by enhancing the efficiency of phosphorus absorption in the intestine and in the kidney. It should be noted that parathyroid hormone has two opposing effects. As noted previously, parathyroid hormone increases urinary phosphate excretion. At the same time, it also increases the synthesis of 1α,25(OH) 2 D 3 by stimulating the activity of the 1α-hydroxylase enzyme in the kidney. In turn, 1α,25(OH) 2 D 3 increases the efficiency of phosphorus absorption in the intestine and kidney. The phosphatonins, in contrast, increase renal phosphate excretion and inhibit 25-hydroxyvitamin D 1α-hydroxylase activity, thereby further decreasing the retention of phosphorus.
Figure 69.2 shows the physiological changes known to occur with low or high dietary intakes of phosphate. A decrease in serum phosphate concentrations results in increased ionized calcium concentrations, decreased parathyroid hormone secretion, and a subsequent decrease in renal phosphate excretion. At the same time, by parathyroid hormone-independent mechanisms, there is an increase in renal 25-hydroxyvitamin D 1α-hydroxylase activity, increased 1α,25(OH) 2 D 3 synthesis, and increased phosphorus absorption in the intestine and reabsorption in the kidney. Conversely, with elevated phosphate intake, there are decreased calcium concentrations, increased parathyroid hormone release from the parathyroid gland, and increased renal phosphate excretion. Increased serum phosphate concentrations inhibit renal 25-hydroxyvitamin D 1α-hydroxylase and decrease 1α,25(OH) 2 D 3 synthesis. Reduced 1α,25(OH) 2 D 3 concentrations decrease intestinal phosphorus absorption as well as renal phosphate reabsorption. All of these factors tend to bring serum phosphate concentrations back into the normal range.
The Physiology of Phosphate in the Kidney
In conditions of phosphate deprivation, the kidney rapidly increases tubular phosphate reabsorption and reduces urinary phosphate excretion to negligible levels in order to preserve phosphate balance. In infants and children, phosphate reabsorption is high so as to maintain a positive phosphate balance required for growth. Conversely, decreased phosphate reabsorption has been demonstrated in the elderly. Phosphate is freely filtered at the glomerulus. Under conditions of normal dietary phosphate intake, and in the presence of intact parathyroid glands, approximately 20% of the filtered phosphate load is excreted. The other 80% of the filtered load of phosphate is reabsorbed by the renal tubules.
The proximal tubules are the major sites of phosphate reabsorption along the nephron. There is little phosphate reabsorption between the late proximal tubule and the early distal tubule in animals with intact parathyroid glands. However, in the absence of parathyroid hormone, phosphate is avidly reabsorbed between the late proximal tubule and early distal tubule, reflecting phosphate reabsorption by the proximal straight tubule ( Fig. 69.3 ). Phosphate transport rates are approximately three times higher in the proximal convoluted than in the proximal straight tubules. Renal phosphate handling is characterized by intranephronal heterogeneity, reflecting segmental differences in phosphate handling within an individual nephron as well as internephronal heterogeneity.
The uptake of phosphate is mediated by sodium-phosphate cotransporters that are located at the apical border of proximal tubule cells (NaPi IIa and NaPi IIc). The structure and physiology of these phosphate transport molecules have been extensively reviewed, and the reader is directed to other publications in this regard. The sodium-phosphate cotransporters are highly homologous and are predicted to have similar structures. Mice with ablation of the NaPi IIa gene exhibit renal phosphate wasting, and it is estimated that the NaPi IIa transporter is responsible for approximately 85% of proximal tubular phosphate transport which contributes to the adaptive increase in tubular phosphate transport in animals fed a low-phosphate diet ( Fig. 69.4 ).
Factors Regulating Renal Phosphate Excretion
Dietary Phosphate and Renal Phosphate Reabsorption
The influence of dietary phosphate intake on the urinary excretion of phosphate has been known for many years. The reabsorption of phosphate is decreased in animals fed a high-phosphate diet, whereas animals with a low intake of phosphate reabsorb almost 100% of the filtered load of phosphate. These changes in phosphate reabsorption are associated with parallel changes in the abundance of NaPi IIa and IIc transporters. The dietary intake of phosphate can differ considerably depending on the ingestion of foods containing varying amounts of phosphate.
Although dietary phosphate deprivation results in marked changes in the plasma concentrations of several hormones ( Fig. 69.2 ) that contribute to the increases in phosphate reabsorption, the enhanced tubular reabsorption can also be demonstrated independent of changes in these hormones. The mechanism of upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after two hours) in the expression of Na/Pi cotransporter, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4–6 hours), resulting in an increase in NaPi-4 mRNA abundance ( Fig. 69.5 ). Although the changes in phosphate reabsorption in response to a low- or high-phosphate meal are demonstrable within two hours, there are not always concomitant alterations in plasma phosphate concentrations. Thus, the sensing mechanism that initiates the renal adaptations in phosphate reabsorption to the changes in phosphate intake is speculative. The enhanced phosphate reabsorption of short-term phosphate deprivation has been linked to decreased intrarenal synthesis of dopamine and/or stimulation of beta adrenoreceptors, since infusion of dopamine or propranolol restores the phosphaturic response to PTH in short-term (less than three days) phosphate deprivation. The concept of central control of phosphate homeostasis was suggested since decreased dietary phosphate intake upregulated the NaPi IIa expression in the brain and increased phosphate intake downregulated the expression of NaPi IIa in the brain. In this study, increasing cerebrospinal fluid phosphate concentrations in the presence of low plasma phosphate concentrations reversed the adaptations to feeding a low-phosphate diet, suggesting that the phosphate concentration in the brain regulates not only central but also renal expression of NaPi IIa transporters. Studies using cultured renal proximal tubular cells provide persuasive evidence of an intrinsic ability of these cultured cells to increase phosphate transport when exposed to a low phosphate concentration in the medium. In addition to the factors that play a role in enhancing or decreasing phosphate reabsorption in the proximal nephron in response to changes in dietary phosphate noted previously, it should be remembered that alterations in serum phosphate concentrations also alter 1α,25(OH) 2 D 3 synthesis and serum concentrations. Infusions of 1α,25(OH) 2 D 3 increase the renal reabsorption phosphate, predominantly in the proximal nephron. Since the proximal tubule is a major site of phosphate reabsorption, it is the primary site for the tubular adaptation to changes in dietary phosphate intake. The enhanced phosphate reabsorption along the nephron during phosphate deprivation in specific nephron subsegments is dependent on the length and severity of phosphate deprivation.
Parathyroid Hormone and Renal Phosphate Reabsorption
Plasma-ionized calcium levels are a critical determinant of PTH secretion. A fall in plasma-ionized calcium increases PTH secretion and an elevation of plasma-ionized calcium above normal levels decreases PTH secretion. Parathyroidectomy decreases renal phosphate excretion and, conversely, injection of PTH increases urinary phosphate excretion. Micropuncture studies show that PTH decreases and parathyroidectomy increases phosphate reabsorption along the proximal tubule ( Fig. 69.3 ). The proximal straight tubule is an important site of PTH modulation of phosphate transport and may be critical in the final regulation of phosphate excretion. Parathyroid hormone maintains phosphate homeostasis as a result of its regulation of the sodium phosphate cotransporters in the kidney. Renal sodium-phosphate cotransporters are reduced in number along the apical borders of proximal tubular cells following the administration of parathyroid hormone 1 through 34 but not by the administration of parathyroid hormone 3 through 34. The renal sodium-phosphate cotransporters NaPi IIa have been shown to be internalized and degraded within the lysosomes. Disruption of the NaPi IIa gene in mice resulted in increased excretion of phosphate compared to wildtype mice and a resistance to the phosphaturic response to PTH, although the cyclic adenosine monophosphate (cAMP) response is normal ( Fig. 69.4 ). Under conditions where the phosphaturic effect of PTH is blunted or absent, such as short-term phosphate deprivation or acute respiratory alkalosis, the inhibitory effect of PTH on phosphate reabsorption by the proximal convoluted tubule remains intact. However, this increased delivery of phosphate is blunted by enhanced reabsorption by the proximal straight tubule. These studies suggest that the regulation of phosphate reabsorption by PTH in the proximal convoluted and proximal straight tubule subsegments may be mediated by different mechanisms. It should be noted that parathyroid hormone has two opposing effects. As noted previously, parathyroid hormone increases urinary phosphate excretion. At the same time, it also increases the synthesis of 1α,25(OH) 2 D 3 by stimulating the activity of the 25-hydroxyvitamin D 3 1α-hydroxylase enzyme in the kidney.
Vitamin D and Renal Phosphate Reabsorption
A complex interrelationship exists between vitamin D and PTH. Both hormones play important roles in calcium and phosphate regulation. Decreases in plasma ionized calcium levels increase PTH levels and PTH also stimulates the renal conversion of 25(OH) 2 D 3 to 1,25(OH) 2 D 3 by the 25-hydroxyvitamin D 3 1α-hydroxylase located in the proximal tubule of the kidney. Dietary phosphate deprivation or hypophosphatemia induces 25-hydroxyvitamin D 3 1α-hydroxylase. Mice or rats, but not pigs, fed a low-phosphate diet show a decrease in the activity of the 25-hydroxyvitamin D 3 24-hydroxylase (a renal enzyme involved in the catabolism of 1,25(OH) 2 D 3 ) compared with rats fed a normal phosphate diet within 24 hours of phosphate restriction. Vitamin D modestly decreases renal phosphate excretion, and its primary effect is to enhance phosphate transport in the intestine. Vitamin D receptor (VDR)-mutant mice exhibit decreased serum phosphate, however, phosphate transport by renal cortical brush border membranes, phosphate excretion or NaPi IIa or NaPi IIc mRNA levels were not different between VDR-null or wildtype mice, while NaPi IIa protein expression and NaPi IIa cotransporter immunoreactive signals were slightly but significantly decreased in the VDR −/− mice compared with the wildtype mice. When VDR knockout mice were fed a low-phosphate diet, serum phosphate concentrations were more markedly decreased in the VDR knockout mice than in the wildtype mice. Other studies performed in vitamin D receptor and 25-hydroxyvitamin D 1α-hydroxylase null mutant mice show that both these knockout mice adapt to phosphate deprivation with increased NaPi IIa protein in a manner similar to that found in wildtype mice. However, when these mice were fed a high-phosphate diet, phosphate excretion was less in the vitamin D receptor and 25-hydroxyvitamin D 1α-hydroxylase null mutant mice compared to the wildtype mice. In vitamin D-deprived rats, NaPi IIa transporter protein and mRNA were reported to be decreased in juxtamedullary but not superficial renal cortical tubules compared with normal rats.
Insulin, Growth Hormone, Insulin-Like Growth Factor, and Renal Phosphate Reabsorption
Insulin decreases plasma phosphate and phosphate excretion in human and animal models. This enhanced renal phosphate reabsorption can be demonstrated in the absence of changes in blood glucose, PTH, and phosphate levels or urinary sodium excretion. Initial micropuncture studies by DeFronzo et al. demonstrate enhanced phosphate reabsorption in hyperinsulinemic dogs. Conversely, somatostatin infusion decreases plasma insulin levels and increases phosphate excretion. Growth hormone decreases phosphate excretion and has been postulated to contribute to increased phosphate reabsorption and positive phosphate balance demonstrated in growing animals. Administration of a growth hormone antagonist for 4 days to immature rats suppressed growth in these rats and was associated with increased phosphate excretion and a decreased transport capacity for phosphate reabsorption. Subsequent studies performed in juvenile rats in which growth hormone was suppressed showed increase phosphate excretion to levels comparable to adult rats as a result of decreased NaPi IIa expression, demonstrating the important role for growth hormone in the enhanced phosphate reabsorption in developing animals. Hammerman et al. demonstrated that growth hormone administration increased phosphate uptake by brush border membrane vesicles prepared from kidneys of adult dogs. These effects of growth hormone on phosphate reabsorption may also be due to insulin-like growth factor-1 (IGF-1). Growth hormone stimulates the renal synthesis and release of IGF-1. The addition of IGF-1 to cultured renal opossum kidney cells stimulates sodium-dependent phosphate transport. A selective increase in sodium-dependent phosphate uptake was detectable after 15 minutes and is maximal at five hours. Chronic administration of IGF-1 infused by osmotic mini-pump for six days significantly increased the maximal tubular reabsorption of phosphate in the presence and absence of PTH and enhanced phosphate transport by renal brush border membranes.
Renal Nerves, Catecholamines, Dopamine, and Serotonin
Acute renal denervation increases urinary phosphate excretion independent of parathyroid hormone. Numerous studies have demonstrated that acute renal denervation or the administration of catecholamines alters phosphate reabsorption. The increase in urinary phosphate excretion after acute renal denervation could be due to both increased production of dopamine and decreased α- or β-adrenoreceptor activity, since acute renal denervation has been shown to initially increase renal dopamine excretion and almost completely abolish norepinephrine and epinephrine levels in the kidney. Epinephrine decreases plasma phosphate, presumably by shifting phosphate from the extracellular into the intracellular space. The hypophosphatemic response to isoproteronol infusion is blocked by propranolol, suggesting involvement of the beta adrenoreceptors. Infusion of isoproteronol markedly enhances renal phosphate reabsorption in normal rats and in hypophosphatemic mice. The enhanced phosphate reabsorption and attenuated phosphaturic response to PTH observed in acute respiratory alkalosis and phosphate deprivation is blocked by infusion of propranolol, suggesting a possible role for stimulation of β-adrenoreceptors in these conditions. Stimulation of α-adrenoreceptors by the addition of epinephrine to cultured opossum kidney cells blunts the PTH-induced increase in cAMP levels and the inhibition of phosphate transport. Stimulation of α2-adrenoreceptors in vivo has also been demonstrated to attenuate the phosphaturic response to PTH. Dopamine infusion and the infusion of L-DOPA or glupopa, dopamine precursors, increase phosphate excretion in the absence of PTH. Dopamine administration has been reported to decrease phosphate transport in cultured opossum kidney cells and rabbit proximal straight tubules. Studies suggest that dopamine may be a proximal tubular paracrine substance in the regulation of phosphate reabsorption. The enzyme that converts L-DOPA to dopamine is located exclusively in the proximal convoluted and straight tubules, also the primary sites of phosphate reabsorption. Increasing dietary phosphate intake increases urinary dopamine excretion and phosphate excretion. Inhibition of endogenous dopamine synthesis by the administration of carbidopa to rats resulted in decreased dopamine and phosphate excretion, suggesting a role for endogenous dopamine in phosphate regulation. A potential paracrine role for dopamine in phosphate regulation was strengthened by studies performed in opossum kidney cells that demonstrated that the addition of dopamine or L-DOPA selectively decreased phosphate uptake ( Fig. 69.6 ). Furthermore, phosphate-replete OK cells produced more dopamine from L-DOPA than phosphate-deprived cells. Administration of dopamine to phosphate-deprived or respiratory alkalotic rats increases phosphate excretion and enhances the phosphaturic response to PTH. Subsequent studies in opossum kidney cells performed by several laboratories demonstrated that increasing dopamine synthesis inhibits phosphate transport by multiple mechanisms including activation of DA1 and DA2 receptors. More recent studies performed using mouse kidney slices, perfused proximal tubules, and opossum kidney cells examined the effect of dopamine on NaPi IIa expression and localization using DA1 and DA2 agonists. In these studies, dopamine induced the internalization of NaPi IIa by activation of luminal DA1 receptors. Renal proximal tubules also synthesize serotonin from 5-hydroxytryptophan by the same enzyme that converts L-DOPA to dopamine. Incubation of opossum kidney cells with either serotonin or 5-hydroxytryptophan enhanced phosphate transport and raises the possibility that serotonin may also be involved in the physiological regulation of renal phosphate transport.
The Phosphatonins and Renal Phosphate Reabsorption
The term “phosphatonin” was introduced to describe a factor or factors responsible for the inhibition of renal phosphate reabsorption and altered 25-hydroxyvitamin D 1α-vitamin D regulation observed in patients with tumor-induced osteomalacia. Cai et al. described a patient with tumor-induced osteomalacia in whom the biochemical characteristics of hypophosphatemia, renal phosphate wasting and reduced 1α,25(OH)2D disappeared following removal of the tumor. A similar biochemical phenotype exists in patients with X-linked hypophosphatemic rickets (XLH) and the animal model, the Hyp mouse. Several investigators have shown the presence of circulating factors in the serum of Hyp mice that inhibit sodium-dependent phosphate transport in the kidney. Further studies demonstrated that patients with the disease, autosomal dominant hypophosphatemic rickets (ADHR), had activating mutations within the fibroblast growth factor homolog, fibroblast growth factor 23 (FGF-23). Its persistence in the circulation resulted in the biochemical phenotype of hypophosphatemia, renal phosphate wasting and low 1α,25(OH) 2 D concentrations. Studies using serial analysis of gene expression (SAGE) identified additional genes that were overexpressed in tumors associated with tumor-induced osteomalacia. Some studies have identified several new factors that also play a role in the regulation of phosphorus transport and homeostasis. These include the “phosphatonins,” FGF-23, and sFRP-4 that induce a state of negative phosphate balance directly by inhibiting renal phosphate reabsorption in the proximal tubule and indirectly by inhibiting the synthesis of 1α,25(OH) 2 D 3 and reducing the intestinal absorption of phosphorus. Two factors, fibroblast growth factor 7 (FGF-7) and MEPE have been shown to inhibit phosphate transport in renal epithelial cells in culture and, in the case of matrix extracellular phosphorus glycoprotein, to induce phosphaturia in mice. FGF-7 and MEPE, however, have not been demonstrated to prevent compensatory increases in serum 1α,25(OH) 2 D 3 concentrations seen in hypophosphatemic states or to directly inhibit 25-hydroxyvitamin D 1α-hydroxylase activity. In contrast, the phosphatonins increase renal phosphate excretion and inhibit 25-hydroxyvitamin D 1α-hydroxylase activity, thereby further decreasing the retention of phosphorus.
Fibroblast Growth Factor-23
As noted previously, FGF-23 was initially postulated to be the factor responsible for autosomal dominant hypophosphatemic rickets. A mutation in the FGF-23 gene resulted in expression of a FGF-23 protein that was resistant to proteolysis and with a prolonged half-life and biopotency. Recombinant FGF-23 produced hypophosphatemia when administered intraperitoneally to mice. Serum calcium concentrations did not change following the administration of the peptide. When Chinese hamster ovary cells were transfected with an FGF-23 expression plasmid and cells were implanted in nude mice, the animals became hypophosphatemic and the fractional excretion of phosphate was increased within 10 days. Alkaline phosphatase concentrations increased in the serum consistent with changes in bone mineralization. Radiologic evidence of rickets in the long bones and histological evidence of rachitic changes were observed after several weeks. There was a decrease in the amount of messenger RNA for the 25-hydroxyvitamin D 1α-hydroxylase. In support of these studies, Bowe et al. demonstrated that recombinant FGF-23 inhibited sodium-dependent phosphate transport in opossum kidney cells. Furthermore, intravenous infusion of recombinant FGF-23 into mice caused a rapid, dose-dependent increase in the fractional excretion of phosphate with little or no change in sodium excretion ( Fig. 69.7 ). These studies suggest that FGF-23 has direct actions on renal phosphate transport. The role of FGF-23 in modulating plasma phosphate concentrations and 25 hydroxyvitamin D 1α-hydroxylase levels was further supported by the generation of FGF-23 null mutant mice. These mice had reduced growth rate and died 10 to 14 weeks after birth. Serum phosphate concentrations were elevated within 10 days after birth and serum calcium concentrations became moderately elevated 2 weeks after birth. Interestingly, these mice developed increased renal 25-hydroxyvitamin D 1α-hydroxylase messenger RNA levels and associated increases in serum 1α,25(OH) 2 D concentrations. A moderate increase in serum calcium concentrations was also observed that could be a consequence of the increased 1α,25(OH) 2 D concentrations and increased intestinal calcium transport. Parathyroid hormone concentrations were diminished in the homozygous mutant mice only at 9 weeks of age. Long bones displayed abnormal mineralization and reduced growth plate. The TmP/GFR was significantly increased in FGF-23 in null mutant animals. Conversely, transgenic mice overexpressing FGF-23 have reduced serum phosphate concentrations, increased phosphate excretion, and reduced renal sodium-phosphate cotransporter, NaPi IIa. In addition to changes in phosphate homeostasis, chronic overexpression of FGF-23 has also been linked to disturbances in vitamin D metabolism, calcium homeostasis and increased PTH levels. The exact interaction and the relative contribution of FGF- 23, PTH, and vitamin D on phosphate homeostasis in these models of chronic FGF-23 excess remains unknown.
The role of FGF-23 in the physiologic regulation of phosphate homeostasis has been addressed by studies to determine the effect of dietary phosphate intake on serum FGF-23 levels. In healthy humans, the effects of phosphate loading and deprivation have shown modest or no changes in circulating FGF-23 concentrations. In one study, an increased phosphate intake slightly increased serum FGF-23. In contrast, other studies in humans did not demonstrate an effect of dietary phosphate intake on serum FGF-23 levels. In rats with renal failure, an increase in dietary phosphate has been shown to increase FGF-23 concentrations in the serum. Future studies are necessary to determine the source of FGF-23 and how its expression is regulated.
The mechanism of action of FGF-23 on phosphate transport is currently unknown. Limited in vitro binding studies suggest that FGF23 may bind to FGFR-Fc fusion proteins. Furthermore, tyrosine kinase inhibitors that are known to inhibit signaling through FGFRs block the effect of FGF-23 on sodium-dependent phosphate opossum kidney cells. These results raise the possibility that FGF-23 may signal through one of the known FGFRs.
Secreted Frizzled Related Protein-4
sFRP-4 was among the most consistently overexpressed genes found associated with oncogenic osteomalacia. To assess whether sFRP-4 has phosphatonin activity, it was expressed by recombinant methods in COS or insect cells. Increasing concentrations of the recombinant protein were added to opossum kidney cells to determine whether it inhibits sodium-dependent phosphate transport. We observed that sFRP-4 inhibited sodium-dependent phosphate transport in opossum kidney cells in a dose-dependent manner at concentrations in the pg/ml range. When infused into rats, sFRP-4 increased renal phosphate excretion at 2 and 8 hours following initiation of the sFRP-4 infusion ( Fig. 69.8 ). Minimal changes in sodium excretion were seen and calcium excretion did not change. Interestingly, the effects of sFRP-4 were also demonstrated in parathyroidectomized rats, thus demonstrating that parathyroid hormone was not essential for the phosphaturic effect of sFRP-4. During an 8-hour intravenous infusion of sFRP-4, serum phosphate concentrations decreased and phosphate excretion increased. However, no change in 25-hydroxyvitamin D 1α-hydroxylase messenger RNA concentrations was noted in the kidney. The infusion of sFRP-4 was associated with a decrease in β-catenin concentrations in renal cells and an increase in phosphorylated β-catenin, thereby demonstrating that sFRP-4 may act as an antagonist against Wnt molecules in the kidney. Additionally, sFRP-4 was detected in the plasma of healthy subjects and in patients with tumor-induced osteomalacia, although the current assay did not detect elevated levels associated with tumor-induced osteomalacia. Thus, the data published to date suggest that sFRP-4 is a phosphatonin. Details concerning the mechanism by which FRP-4s inhibits renal phosphate reabsorption and its relationship to FGF-23 will need to be elucidated in the future. Studies performed in mice in which the sFRP-1 gene was deleted demonstrate that these mice exhibit enhanced trabecular bone formation in adult mice. In these studies, plasma phosphate concentrations were significantly increased by 29% in male mice at 18 to 20 weeks of age, consistent with the possibility that sFRP proteins modulate renal phosphate reabsorption.
Matrix Extracellular Phosphoglycoprotein
Matrix extracellular phosphoglycoprotein (MEPE) is also among the most abundantly overexpressed mRNA species found in tumors associated with renal phosphate wasting and osteomalacia. Recently, MEPE has been expressed in insect cells and administered to mice in vivo . The protein causes renal phosphate wasting and a reduction in serum phosphate concentrations in vivo . Additionally, there is inhibition of sodium-dependent phosphate uptake noted in opossum kidney cells exposed to the protein. MEPE also inhibits bone mineralization in vitro and MEPE null mice have increased bone mineralization. This suggests that it may play a role in the pathogenesis of X-linked hypophosphatemic rickets in which there is phosphate wasting and evidence for a mineralization defect that is independent of low phosphate concentrations in the extracellular fluid. Recent evidence suggests that concentrations of this substance are increased in the serum of patients with X-linked hypophosphatemic rickets. It has been suggested that MEPE is a substrate for PHEX and that PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE-ASARM peptide, an inhibitor of mineralization (minhibin). Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif. The ASARM peptide is believed to inhibit mineralization in vivo . The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in Hyp. MEPE concentrations have been measured in normal humans and concentrations of the protein appear to correlate with bone mineral density and serum phosphate concentrations.
Fibroblast Growth Factor 7
A recent report has shown that FGF-7 is overexpressed in tumors associated with osteomalacia and renal phosphate wasting. FGF-7 protein inhibited sodium-dependent phosphate transport in opossum kidney cells. Anti–FGF-7 antibodies attenuated the inhibitory effect of tumor supernatants on sodium-dependent phosphate transport. Only low concentrations of FGF-23 were present in the supernatant medium of tumor cells. At present it is not known if FGF-7 circulates in plasma, whether it alters 25-hydroxyvitamin D 1α-hydroxylase levels or whether it is elevated in the plasma of subjects with tumor induced osteomalacia. Nevertheless, the report does point to the complexity of factors involved in the pathogenesis of tumor induced osteomalacia.
Acute and Chronic Hypophosphatemia
Hypophosphatemia is a commonly seen condition in the hospital setting. By definition, this is a decrease in serum phosphate concentrations below those seen in a normal healthy population. Serum phosphate concentrations vary with age, being higher in young individuals than in adults. Table 69.1 shows concentrations of serum phosphorus in males and females of different ages as determined at the Mayo Clinic.
|Age, y||Concentration, mg/dl||Age, y||Concentration, mg/dl|
Hypophosphatemia does not necessarily imply an overall deficiency of phosphate, since serum phosphate concentrations can change as a result of redistribution of phosphate from serum and extracellular fluid into bone and soft tissues. When the underlying disorder causing the redistribution resolves, serum phosphate concentrations returned to normal. Hypophosphatemia can be the result of decreased intake or absorption of phosphorus, a redistribution of phosphate from the serum into cells, or decreased renal phosphate reabsorption. Table 69.2 shows the causes and mechanisms of hypophosphatemia. Severe hypophosphatemia, often requiring therapy with phosphate, occurs in the following conditions: acute renal failure (especially when there has been significant tissue damage and necrosis), chronic alcoholism and alcohol withdrawal, dietary deficiency and therapy with phosphate-binding antacids, hyperalimentation, neuroleptic malignant syndrome, recovery from diabetic ketoacidosis, recovery from exhaustive exercise, after kidney transplantation, respiratory alkalosis, severe thermal burns, therapeutic hyperthermia, Reye syndrome, after major surgery, periodic paralysis, acute malaria, drug therapy with ifosamide or cisplatin, acetaminophen intoxication, or cytokine infusions with tumor necrosis factor or interleukin-2.
|Pseudohypophosphatemia||Cellular Uptake Syndromes|
|Mannitol||Recovery from hypothermia|
|Acute leukemia||Histiocytic lymphoma|
|Acute myelomonocytic leukemia|
|Decreased dietary intake||Acute myelogenous leukemia|
|Chronic myelogenous leukemia in blast crisis|
|Decreased intestinal absorption||Treatment of pernicious anemia|
|Vitamin D deficiency||Erythropoietin therapy|
|Steatorrhea||Hungry bone syndrome|
|Secretory diarrhea||After parathyroidectomy|
|PO43− binding antacids|
|Increased excretion into the urine|
|Shift from serum into cells||Hyperparathyroidism|
|Respiratory alkalosis||Renal tubule defects|
|Heat stroke||X-linked hypophosphatemic rickets|
|Neuroleptic malignant syndrome||Hereditary hypophosphatemic rickets with hypercalciuria|
|Hepatic coma||Polyostotic fibrous dysplasia|
|Panic attacks||Oncogenic osteomalacia|
|Psychiatric depression||Recovery from hemolytic-uremic syndrome|
|Hormonal effects||Licorice ingestion|
|Glucagon||Inappropriate secretion of antidiuretic hormone|
|Anovulatory hormones||Aminophylline therapy|
Hypophosphatemia can cause a variety of clinical disorders. Red cell, leukocyte, and platelet dysfunction has been described. In red cells, hypophosphatemia is associated with a decline in 2,3-diphosphoglycerate and adenosine triphosphate. Structural defects in the red cell resulting in hemolysis have been described. In severe hypophosphatemia, leukocyte chemotaxis, phagocytosis, and bactericidal activity are depressed. Platelet dysfunction manifests by thrombocytopenia, an increase in platelet diameter, accelerated platelet disappearance, impaired clot retraction, and hemorrhage into the skin and intestine has been described. Central nervous system dysfunction has been described in patients with severe hypophosphatemia. Patients may manifest irritability, weakness, numbness, paresthesias, confusion, obtundation, seizures and coma. Rhabdomyolysis has been described in severe hypophosphatemia, especially in the context of alcoholism. Myofibrillar damage has been observed in severe hypophosphatemia. A reversible cardiomyopathy has been described in experimental animals and patients with severe hypophosphatemia. Also, dysfunction can also cause weakness of the diaphragm and has, in some instances, been described to result in respiratory insufficiency. In some instances, dietary phosphorus deprivation and severe hypophosphatemia have been associated with an osteolytic syndrome. A number of other less well-defined abnormalities have been described in patients with hypophosphatemia, but the precise cause-and-effect relationship remains to be firmly established.
Pathophysiology of Hypophosphatemia in Clinical Disorders
An examination of the pathophysiology of hypophosphatemia in various clinical disorders is instructive because it brings to light various mechanisms that are normally involved in the regulation of serum phosphate concentrations. This is particularly the case in the emerging area of inherited rickets and acquired forms of rickets due to tumors. Here, new substances have been isolated and identified that play an important role in the pathophysiology of the syndromes and that might also play an important role in the regulation of phosphate concentrations under normal circumstances.
In the clinical setting, the most common cause of acute hypophosphatemia is respiratory alkalosis (see previous sections), which causes a rapid redistribution of phosphate from the serum into the intracellular space, resulting in a marked decrease in plasma phosphate ( Fig. 69.9 ). Since respiratory alkalosis decreases the plasma phosphate concentration, it is likely that changes in the filtered load of phosphate contribute to the changes in phosphate excretion. However, a direct effect of respiratory alkalosis changes on renal phosphate reabsorption has also been demonstrated in the absence of changes in the filtered load of phosphate. The effects of acute respiratory alkalosis on plasma phosphate and on phosphate reabsorption are due to the decrease in PCO 2 rather than the concomitant changes in blood pH. The effects of respiratory alkalosis on phosphate homeostasis are mediated, in part, by release of catecholamines.
Sepsis is associated with hypophosphatemia. In septic patients with hypophosphatemia, 80% of these patients had very high levels of tumor necrosis factor (TNF) and interleukin-6. Injection of various interleukins and TNFα in experimental animals and in humans decreased plasma phosphate, suggesting a role for inflammatory cytokines in the hypophosphatemic effect of sepsis.
The earliest descriptions of hypophosphatemia with refeeding were described in starved patients in war time. This syndrome is frequently observed in clinical settings in patients after prolonged fasting, massive weight loss after gastric surgery, chronic alcoholism, chronic malnutrition, oncology patients, and anorexia nervosa. Hypophosphatemia is observed after either parenteral or enteral refeeding and is mediated by changes in carbohydrate metabolism, decreased insulin concentrations and increased glucagon levels.
Hypophosphatemia commonly occurs in the context of alcohol withdrawal. It affects approximately 50% of patients with alcoholism, requiring hospitalization. The etiology of hypophosphatemia is multifactorial in patients admitted for alcoholism. Poor nutritional intake, magnesium deficiency which can result in hypophosphatemia as a result of renal phosphate wasting and alcohol-induced abnormalities in tubular function probably all contribute to the pathogenesis of hypophosphatemia in such patients. Abstinence from alcohol is generally associated with the recovery from hypophosphatemia.
Hypophosphatemia has frequently been reported in transplant patients with concomitant decreased bone mineral density. This hypophosphatemia is the result of decreased renal reabsorption of phosphate and is thought to be due, in part, to elevated PTH levels as a result of prior renal failure and the administration of glucocorticoids. However, some transplant patients exhibit increased phosphate excretion even when kidney function and PTH levels are normal. A recent study examined the mechanism and time course of the post-transplant hypophosphatemia. In this study, early (two weeks to one month) transplantation patients exhibited hypophosphatemia and phosphaturia, compared with control subjects or late (9–12 months) post-transplantation patients. Serum from these patients with hypophosphatemia inhibited phosphate transport by opossum kidney cells, suggesting the presence of a circulating phosphatonin substance. Serum FGF-23 levels are known to be markedly increased in patients with chronic renal failure; however, they are not significantly increased following renal transplantation.
This syndrome is associated with chronic hypophosphatemia and osteomalacia that is corrected following removal of the tumor. Initial experiments performed in patients with tumor-induced osteomalacia (TIO) were the first to describe “phosphatonins” as a circulating factor from the serum of these patients that inhibits phosphate transport in opossum kidney cells. Numerous reports now show that serum FGF-23 is elevated in some, but not all, patients with oncogenic or tumor-induced osteomalacia. Removal of the tumor is generally associated with a reduction in serum FGF-23 concentrations, and there is a temporal association between the reduction in serum FGF-23 concentrations, the increase in serum phosphate concentrations, a decrease in renal phosphate wasting and an increase in serum 1α,25(OH) 2 D 3 concentrations. In some patients, FGF-23 concentrations are not greatly increased and do not dramatically decrease following tumor removal. The increase in serum FGF-23 concentrations is consistent with the overexpression of messenger RNA and protein for FGF-23 within the tumors themselves. Recently, venous sampling has demonstrated a gradient between FGF-23 concentrations in tumor venous effluent and FGF-23 concentrations in peripheral blood, suggesting that elevations of serum FGF-23 are due to direct secretion from the tumors. As described previously, several phosphatonin molecules, including secreted frizzled related protein-4, MEPE, and fibroblast growth factor-7, have all been isolated from tumors from patients with TIO. Increased serum or renal concentrations of these proteins result in decreased renal phosphate reabsorption which contributes to the subsequent hypophosphatemia and osteomalacia.
Autosomal Dominant Hypophosphatemic Rickets
Autosomal dominant hypophosphatemic rickets (ADHR) is an inherited disorder of phosphate homeostasis characterized by decreased renal phosphate reabsorption and hypophosphatemia linked to chromosome 12p13. These biochemical abnormalities are associated with bone pain, muscle weakness, poor growth, and evidence of rickets and osteomalacia. Individuals with this disease present with renal phosphate wasting and inappropriately normal serum 1α,25(OH) 2 D concentrations. As noted earlier, mutations in the FGF-23 gene within a pro-convertase processing site were identified as the cause for autosomal dominant hypophosphatemic rickets. These mutations appear to prevent processing of FGF-23 in mice and enhances the in vivo biological potency resulting in increased serum concentration of FGF-23, decreased serum phosphate concentrations and low 1α,25(OH) 2 D concentrations. When transgenic mice expressing human FGF-23 were generated, these mice exhibited hypophosphatemic rickets suggesting that increased expression of FGF-23 may play a role in this disease syndrome.
X-Linked Hypophosphatemic Rickets
X-linked hypophosphatemic rickets (XLH) is the most common inherited cause of rickets. XLH (and murine model of this diseases, Hyp mouse and the Gy mouse) is an inherited X-linked dominant disorder characterized by hypophosphatemia, normocalcemia and an inappropriately low concentration of serum 1α,25(OH) 2 D. Many studies have demonstrated that in XLH there is decreased renal phosphate reabsorption. This defect in phosphate reabsorption was demonstrated to be due to a humoral factor, since parabiosis performed between a normal and Hyp mouse causes hypophosphatemia in the normal mouse, and this effect is reversible. These results are further supported by cross renal transplantation experiments. When a kidney from a Hyp mouse is transplanted into a normal mouse, phosphate wasting is no longer observed. Conversely, transplantation of a kidney from a normal mouse into a Hyp mouse results in phosphate wasting by the normal transplanted kidney. These data are consistent with a circulating hypophosphatemic substance in the circulation of Hyp mice. The addition of Hyp mouse serum to primary renal cells selectively inhibits phosphate uptake, and the Hyp serum inhibits phosphate transport to a greater extent than normal serum, indicating the presence of a factor in Hyp mouse serum that inhibits phosphate uptake. Positional cloning efforts have shown that PHEX (phosphate regulating endopeptidase on the X-chromosome) is the mutant gene responsible for XLH. The same gene is mutated in mice with the Hyp and Gy mutations (in the Gy mouse there is a deletion of the adjacent spermine gene as well). Since the mutant gene in this syndrome is a protease, it has been thought that a lack of proteolytic activity results in comparing degradation of a hypophosphatemic substance. With the identification of FGF-23 as a hypophosphatemic peptide responsible for phosphate wasting in patients with autosomal dominant hypophosphatemic rickets, and the elevation of FGF-23 in some patients with tumor-induced osteomalacia, considerable effort has been expended to determine whether FGF-23 concentrations are elevated in patients with XLH and whether FGF-23 is a substrate for PHEX. The evidence for elevated FGF-23 expression in patients with X-linked hypophosphatemic rickets is conflicting. Jonsson and coworkers describe elevated serum FGF-23 concentrations in many subjects, whereas Weber and colleagues reported only modestly increased or normal concentrations of FGF-23. In the Hyp mouse, preliminary reports suggest that FGF-23 serum concentrations are elevated and neutralization of FGF-23 with an antibody ameliorates hypophosphatemia and rickets. These data are consistent with the premise that PHEX processes FGF-23 either directly or indirectly under normal circumstances. The inactivation of FGF-23 by PHEX has been demonstrated by some, but not all investigators. Inactivation of PHEX, as occurs in individuals with XLH and in Hyp mice, would reduce FGF-23 degradation and cause renal phosphate wasting and hypophosphatemia.
Fibrous Dysplasia/McCune-Albright Syndrome
Fibrous dysplasia is a genetic, noninherited disease caused by somatic activating missense mutations of GNAS1 that affects the skeleton or with endocrine and cutaneous abnormalities, McCune-Albright syndrome. Phosphate wasting is observed in approximately 50% of these patients and is associated with defects in bone mineralization. Riminucci and coworkers examined the concentrations of FGF-23 in the plasma of patients with fibrous dysplasia, some of whom have hypophosphatemia. These investigators demonstrated that those patients who had low phosphate concentrations had elevated FGF-23 concentrations in the blood, whereas those who had normal phosphate concentrations did not have elevations in FGF-23.
Humoral Hypercalcemia of Malignancy and Hyperparathyroidism
Patients with humoral hypercalcemia of malignancy or primary hyperparathyroidism have elevated serum calcium concentrations, hypophosphatemia, and altered serum 1α,25(OH)D 3 concentrations. Of note, in patients with primary hyperparathyroidism, 1α,25(OH)D 3 concentrations are increased. Whereas, in patients with humoral hypercalcemia of malignancy serum, 1α,25(OH)D 3 concentrations are inappropriately low for the degree of hypophosphatemia that these patients manifest. It is possible that increased circulating FGF-23 could contribute to the hypophosphatemia seen in individuals with these disorders. Recently, serum FGF-23 has been shown to be elevated five- to 10-fold in patients with humoral hypercalcemia of malignancy. Interestingly, the elevations in FGF-23 are not correlated with the concentrations of serum phosphorus. It is possible that a low serum 1α,25(OH)D3 concentration seen in the patients with humoral hypercalcemia of malignancy could have been due to the increased FGF-23 concentrations. Patients with primary hyperparathyroidism have slightly elevated FGF-23 concentrations that do not change after parathyroidectomy.