Clinical Disorders of Phosphate Homeostasis

C H A P T E R

63 Clinical Disorders of Phosphate Homeostasis Karen E. Hansen 1, Marc K. Drezner 2 1

2

University of Wisconsin, Madison, WI, USA William H. Middleton Veterans Administration Medical Center, Madison, WI, USA

INTRODUCTION Extensive studies over the past several decades have established that phosphate homeostasis and vitamin D metabolism are reciprocally regulated. As discussed in Chapters 19 and 34 calcitriol promotes phosphate absorption from the intestine, mobilization from bone, and reabsorption in the renal tubule [1]. In turn, phosphate depletion or hypophosphatemia stimulates renal production of 1,25(OH)2D (calcitriol), while phosphate overload or hyperphosphatemia inhibits renal 25(OH) D-1a-hydroxylase (1a-hydroxylase) activity [2]. Since phosphorus is one of the most abundant constituents of all tissues, disturbances in phosphate homeostasis can affect almost any organ system [3]. Indeed, a deficiency or excess of this mineral can have profound effects on a variety of tissues. Consequences of hypophosphatemia include osteomalacia, rickets, red cell dysfunction, rhabdomyolysis, metabolic acidosis, and cardiomyopathy. By contrast, hyperphosphatemia may lead to soft tissue calcification, hypocalcemia, tetany, and secondary hyperparathyroidism. In many cases, the interrelationship between phosphate homeostasis and vitamin D metabolism precludes establishing whether the consequences of hypo- or hyperphosphatemia are singularly related to this abnormality or are modified by changes in calcitriol production. Occasionally, however, discrimination between these possibilities has been achieved by evaluation of the therapeutic response to phosphate supplementation or depletion. Such studies indicate that few of the phosphate homeostatic disorders respond adequately to therapeutically induced alterations in phosphate alone, but do regress upon coincident modification of the vitamin D status. The detailed control mechanisms that regulate phosphate homeostasis and vitamin D metabolism in

Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10063-0

intestine and kidney are reviewed in Chapters 19 and 26 and overall physiology in Chapter 34. The following is a summary of important elements of the phosphate homeostatic schema and the regulation of vitamin D metabolism that pertain to an understanding of the diseases described in the remainder of this chapter and in Chapter 42.

Regulation of Phosphate Homeostasis Most phosphate (85%) resides in bone, where it associates with calcium; 14% of phosphate is in cells, as a component of lipids, proteins, nucleic acids, and small molecules of metabolic and signaling pathways. Phosphate in serum and extracellular fluids accounts only for 1% of total-body phosphate, but the serum phosphate concentration correlates, in most circumstances, with total-body phosphate content. The kidney is the major arbiter of extracellular phosphate (Pi) homeostasis and plays a key role in bone mineralization and growth. Most of the filtered phosphorus is reabsorbed in the proximal tubule, with approximately 60% of the filtered load reclaimed in the proximal convoluted tubule and 15e20% in the proximal straight tubule [4]. In addition, a small but variable portion (<10%) of filtered phosphorus is reabsorbed in more distal segments of the nephron. The reabsorption of phosphate filtered by the glomerulus is an active, hormonally regulated process and the amount of phosphate reabsorbed is a key determinant of the serum phosphorus concentration [5]. Transepithelial phosphorus transport is effectively unidirectional and includes uptake at the brush border membrane of the renal tubule cell, translocation across the cell and efflux at the basolateral membrane [6]. Apical sodium-dependent phosphate (Na/Pi)

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cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. The uptake at this site is mediated by Naþ-dependent phosphate transporters that reside in the brush border membrane and depend on the Naþ, Kþ-ATPase to maintain the Naþ gradient that drives the transport system [7]. In contrast, basolateral Pi-transport systems are not well defined. Efflux of Pi across the basolateral membrane may involve an anion exchange mechanism and/or a “Pi leak” to complete transcellular reabsorptive flux, and an Naþ-dependent Pi uptake mechanism to guarantee Pi uptake from the interstitium if apical influx is insufficient to maintain cellular metabolism. There are three types of sodium-phosphate cotransporters in the renal proximal tubule cells, all of which have the capacity to induce an increase in Na-dependent Pi uptake in heterologous expression systems [8]. The type 1 cotransporter (NPT1) is an anion carrier that does not specifically mediate phosphate transport and does not contribute significantly to regulation of proximal tubular Pi flux [9,10]. Indeed, heterologous expression studies indicate that the type-I-mediated Na/Pi cotransport does not have the characteristics and regulatory features of brush border membrane Na/Pi cotransport. Rather, these studies suggest that the type I cotransporter has a channel function, mediating the flux of chloride and organic anions [8,10,11]. The type 2 cotransporter family includes three carriers: NPT2a (encoded by the SLC34A1 gene), NPT2b (encoded by the SLC34A2 gene), and NPT2c (encoded by the SLC34A3 gene). Type 3 consists of two transporters: sodium-dependent phosphate transporter 1 (PiT1, encoded by the SLC20A1 gene) and sodium-dependent phosphate transporter 2 (PiT2, encoded by the SLC20A2 gene). NPT2a, which plays a key role in renal Pi reabsorption and phosphate homeostasis, is located almost exclusively in the apical membrane of renal proximal tubular cells [8]. Indeed, studies in adult animals support the vital function of the type 2a receptor in several ways: [1] loss of 70e80% of brush border membrane Na/Pi cotransport upon disruption of the gene encoding the NPT2a [12]; (2) correlation of NPT2a protein abundance in brush border membranes with Na/Pi cotransport activity under a variety of physiological/pathophysiological conditions [8]; and (3) disruption of the NPT2a gene in mice increases urinary phosphate excretion and causes marked hypophosphatemia. The NPT2b transporter is expressed predominantly in the lungs and the small intestine and only weakly in the kidney [13]. After birth, disruption of the gene leads to decreased gastrointestinal phosphate absorption. However, the serum phosphorus concentration remains normal due to a compensatory increase in renal NPT2a.

NPT2c, located at the renal brush border of the proximal tubule, when mutated in humans results in hypophosphatemia, rickets, and hypercalcuria, the HHRH syndrome. In contrast, mice null for Npt2c exhibit no renal phosphate wasting when fed a normal phosphorus diet, suggesting that NPT2a is the major player in renal phosphate transport. However, Segawa et al. [14] demonstrated that mice null for both Npt2a and Npt2c had more severe phosphate wasting and subsequent rickets than mice null for Npt2a or Npt2c alone, indicating a synergistic role of both transporters in phosphate balance. The type 3 Na/Pi cotransporters are cell-surface receptors, first identified as receptors for retroviruses [15], and appear to exhibit ubiquitous renal and extrarenal expression. Although type 3 mRNA expression is detected in all nephron segments [7], studies to localize the type 3 protein in apical or basolateral cell membranes are lacking. However, the type 3 Na/Pi cotransporters may be responsible for basolateral Pi influx in all tubule cells to maintain cell metabolism, as well as in proximal tubule cells under conditions of limited apical influx. Proximal tubular Na/Pi cotransport is regulated by a variety of hormones/metabolic factors, which elicit an increase or decrease in Pi reabsorption through alteration of the NPT2a and NPT2c protein availability. Parathyroid hormone (PTH)-dependent inhibition of Naþ-Pi cotransport depends upon internalization of the cell surface NPT2a protein [16]. In contrast, FGF23 alters renal phosphate reabsorption by altering expression of NPT2a and NPT2c. FGF23 binds predominantly to the renal FGFR1 receptor through a process that requires membrane-bound protein, klotho, located in the distal tubule. Interaction of FGF23 with the heteromeric FGFR1/Klotho receptor elicits MAPK signaling in the distal convoluted tubule, which likely downregulates NPT2a and NPT2c expression in the proximal convoluted tubules by an ill-defined paracine process [17]. Acute renal adaptation to phosphate restriction is associated with an increase in NPT2a protein, which is rapidly reversed by a high-Pi diet [18]. Not surprisingly, NPT2a protein availability is also central to modulation of the abnormal renal phosphate transport underlying a large number of phosphate homeostatic disorders. These changes in Na/Pi cotransport and NPT2a protein expression occur predominantly in the absence of variations in NPT2a mRNA levels. Thus, rapid increments or decrements in Na/Pi cotransport follow either membrane insertion of NPT2a protein, which may be preceded by de novo synthesis of the protein, or membrane retrieval of NPT2a protein, followed by lysosomal degradation [8]. Although such membrane trafficking of NPT2a protein is an unequivocally central element in the regulation of Na/Pi cotransport, changes

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INTRODUCTION

Phosphate-dependent Modulation of Vitamin D Metabolism The production of 1,25(OH)2D, the active metabolite of vitamin D, is under stringent control (see Chapter 3). Indeed, 1a-hydroxylation represents the most important regulatory mechanism in the metabolism of vitamin D [24]. In normal adults, serum 1,25(OH)2D concentrations change little in response to repeated dosing with vitamin D, and remain normal, or even decline, in vitamin D intoxication. Phosphorus is one of the three major factors that regulate the activity of the 1a-hydroxylase enzyme [25]. Phosphate depletion and resultant hypophosphatemia stimulate 1a-hydroxylase activity and increase the serum 1,25(OH)2D concentration, whereas phosphate loading and consequent hyperphosphatemia inhibit formation of this metabolite. The mechanism whereby phosphorus modulates this adaptive effect remains unknown. Regardless of the mechanism,

the potential role of phosphorus in regulating vitamin D metabolism is central to understanding and appropriately treating many of the clinical disorders of phosphate homeostasis. As will become evident in the remainder of this chapter, however, the aberrant regulation of vitamin D metabolism encountered in many of these diseases is considered paradoxical. Thus, in virtually all disorders secondary to a primary abnormality in renal phosphate transport, hypophosphatemia or hyperphosphatemia is perplexingly associated with decreased or increased serum 1,25(OH)2D levels, respectively. These observations suggest that the effect of phosphorus on 1a-hydroxylase activity may, in fact, occur indirectly and secondary to alterations in renal phosphate transport systems. In this regard, phosphate depletion and hypophosphatemia and phosphate loading and hyperphosphatemia are associated with compensatory changes in renal phosphate transport that may mediate changes in 1,25(OH)2D production. In accord, the prevailing serum calcitriol levels in normal patients and in patients with disorders of renal phosphate transport, X-linked hypophosphatemia (XLH), tumor-induced osteomalacia (TIO), and tumoral calcinosis (TC) display a highly significant positive correlation with the renal tubular maximum phosphate reabsorption per liter of glomerular filtrate (TmP/GFR) [26], supporting the possibility that renal tubular reabsorption of phosphate may be a major determinant of renal 1,25(OH)2D production (Fig. 63.1). Of course, these data do not

84 72 Plasma 1.25(OH)2D (pg/ml)

in NPT2a mRNA levels do occur after prolonged treatment with 1,25(OH)2D, PTH, FGF23 or thyroid hormone, following chronic changes in dietary Pi and in disorders of Pi homeostasis, such as X-linked hypophosphatemia (XLH). Thus, a secondary mechanism operates under select conditions to regulate Na/Pi cotransport. The intracellular signaling mechanisms involved in insertion/retrieval of NPT2a protein remain incompletely understood. However, several studies have identified numerous signals for NPT2a internalization. PTH effects on NPT2a protein are initiated by binding to receptors that activate protein kinase C on apical membranes and protein kinase A and C on basolateral membranes [19]. In contrast, atrial natriuretic proteininduced internalization of NPT2a protein is mediated by protein kinase G activation [20]. Additional investigations suggest that these different signaling pathways converge on the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) pathway to internalize the NPT2a protein [21]. In any case, the signaling process regulating internalization/ retrieval of NPT2a is insufficient to explain the integrated regulation of Pi homeostasis at the kidney. Rather it appears that protein/protein interactions may also participate in regulation of Pi reabsorption. In this regard, NaPi-Cap 1 and NHERF-1, brush border membrane proteins, may interact with NPT2a via one of multiple PDZ-domains, resulting in release of the protein from an apical scaffold, thereby permitting internalization [22,23]. Clearly, further studies are necessary to determine the interrelated mechanisms regulating the many processes that affect the abundance of NPT2a in brush border membranes.

60 XLH TIO Normals Tumoral calcinosis

48 36

r = 0.85 p<0.001

24 12

0

1

2

3

4 7 5 6 TmP/GFR (mg/dl)

8

9

10

FIGURE 63.1 Correlation between renal TmP/GFR and plasma 1,25(OH)2D levels in normals and patients with XLH, TIO, and tumoral calcinosis. Significant linear correlation is evident, suggesting a potential relationship between renal phosphate transport and the prevailing plasma levels of active vitamin D. TIO, tumor-induced osteomalacia. From [29].

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10 8 mg/dl

*

*

6 4 2 0

Control Dietary Control Renal –P –P

Renal 25(OH)D-1-hydroxylase activity

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14 (fmoles/mg kidney/min)

Serum phosphorus concentration

(A)

1,25-Dihydroxyvitamin D produced

63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS

12 10 8 6 4 2 0

Control Dietary Control Renal –P –P

*Significantly different from control at p,0.05

Renal 25(OH)D-1-hydroxylase activity

Serum phosphorus concentration

(B) 10

4 2

1,25 OH2D produced

**

(fmoles/mg kidney/min)

mg/dl

** 6

0 Dietary- P PFA- P

**

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FIGURE 63.2 (A) While hypophosphatemia resulting from phosphate restriction increases renal 25(OH)D-1a-hydroxylase activity, hypophosphatemia of similar magnitude secondary to phosphonoformic acid (PFA)-mediated renal phosphate wasting has no effect on enzyme function. These data suggest that hypophosphatemia is not the proximal trigger mechanism for 1,25(OH)2D production. (B) In similar experiments the effects of phosphate depletion and consequent hypophosphatemia on renal 25(OH)D-1a-hydroxylase activity are blocked by the coincident administration of phosphonoformic acid, which prevents the expected compensatory change in renal phosphate flux attendant upon the depletion. These observations reaffirm the potentially important role of renal phosphate transport in modulating the effects of phosphate for 1,25(OH)2D production.

10 8 6 4 2

0 0

0

**Significantly different from respective control (0,0) at p<0.01

0 Dietary- P PFA- P

0 0

0

PFA, Phosphonofomic acid -P, Phosphate depletion

establish that alterations in 1a-hydroxylase activity, in response to phosphate depletion or loading, are dependent upon renal phosphate transport. However, the observations that phosphate depletion does not increase 1,25(OH)2D production in mice subjected to treatment with phosphonoformic acid, which precludes a compensatory alteration in TmP/GFR, suggests an important role for the renal phosphate transport system in modulating phosphorus-mediated effects on 1a-hydroxylase activity under all conditions (Fig. 63.2). Additionally, preliminary studies have documented that mice with targeted disruption of the NHERF-1 gene, which singularly promotes internalization of proximal tubule Npt2a, manifest ~50% decreased apical membrane localized Npt2a and consequent renal P wasting and hypophosphatemia [22], and paradoxically exhibit inappropriately normal serum 1,25(OH)2D levels, similar to Hyp mice (the murine homolog of XLH) with comparable Npt2a deficiency. In contrast, Tenenhouse et al. [27] concluded from studies in the Npt2ae/e mouse that

Npt2a and renal P transport do not influence 25(OH)D-1a-hydroxylase activity. However, these data may have limited applicability to understanding the role of renal phosphate transport on vitamin D metabolism, since it is well known that compensatory developmental changes often confound studies in knockout mice with complete absence of a protein function. In this regard, several investigators [28,29] reported that the sodium-dependent P cotransporter, Npt2c, normally expressed in murine kidneys of young animals, has sustained activity in adult Npt2ae/e mice, which is likely responsible for the “inappropriate” residual renal P transport in these mutants and the resultant limited hypophosphatemia. The absence of similar Npt2c expression in the kidneys of adult normal and NHERF knockout mice [30] substantiates that enhanced Npt2c expression in adult Npt2ae/e mice may be a unique compensatory mechanism, which limits the applicability of studies in this model to conclusions regarding regulation of vitamin D metabolism.

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While these data indicate that the renal production of 1,25(OH)2D and the renal tubular reabsorption of phosphate are linked, the precise mechanism(s) underlying this association remain uncertain. However, it is clear that understanding the pathophysiology of, and defining appropriate treatment regimens for, many clinical disorders of phosphate homeostasis require investigation of the vitamin D regulatory system.

TABLE 63.1

Disorders of Phosphate Homeostasis

ABNORMAL RENAL PHOSPHATE TRANSPORT Hypophosphatemic syndromes Genetic diseases X-Linked hypophosphatemia (XLH) Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) Tumor-induced osteomalacia (TIO) Autosomal dominant hypophosphatemic rickets (ADHR)

DISORDERS OF PHOSPHATE HOMEOSTASIS

Autosomal recessive hypophosphatemic rickets (ARHR) McCune Albright syndrome (MAS)

The variety of diseases, therapeutic agents, and physiological states that affect phosphate homeostasis are numerous and reflect a diverse pathophysiology. Indeed, rational choice of an appropriate treatment for many of these disorders depends on determining the precise cause for the abnormality. In general, defects in phosphate homeostasis result from impaired renal tubular phosphate reabsorption and a consequent change in the TmP/GFR, an altered phosphate load due to varied intake, or abnormal gastrointestinal absorption or translocation of phosphorus between the extracellular fluid and tissues (Table 63.1). The disorders of renal phosphate transport are the most common of these diseases and have been intensively studied. Indeed, investigation of these conditions has provided new insight into the regulation of renal phosphate transport and the reciprocal regulation of phosphate homeostasis and vitamin D metabolism. In the remainder of this chapter, several clinical states that represent disorders of phosphate homeostasis will be discussed and the potential role of the vitamin D endocrine system in their pathogenesis and phenotypic presentation highlighted.

Fanconi syndrome, type I (FS I) Familial idiopathic Cystinosis (Lignac-Fanconi disease) Oculocerebrorenal (Lowe) syndrome Glycogen storage disease Wilson disease Galactosemia Tyrosinemia Hereditary fructose intolerance Neurofibromatosis Linear nevus sebaceous syndrome Fanconi syndrome, type II (FS II) Acquired disorders Tumor-induced osteomalacia Mesenchymal, epidermal and endodermal tumors Light chain nephropathy Renal transplantation Multiple myeloma Cadmium intoxication

Disorders of Renal Phosphate Transport: Hypophosphatemic Diseases

Lead intoxication Tetracycline (outdated) administration

The disorders of renal phosphate transport, which lead to phosphate wasting and hypophosphatemia, are by far the most common disturbances of phosphate homeostasis. However, the pathophysiological basis of these disorders has remained elusive, largely because the hormonal/metabolic control of phosphate homeostasis at the kidneys and in bone is not completely understood. In this regard, the function of the PTHevitamin D axis is not sufficient to explain the physiological complexity of systemic phosphate homeostasis. However, researchers have identified a class of circulating factors that promote renal phosphorus wasting in patients with TIO, XLH, autosomal recessive hypophosphatemic rickets (ARHR), and autosomal

Hyperphosphatemic syndromes Tumoral calcinosis ALTERED PHOSPHATE LOAD Hypophosphatemic syndromes Decreased phosphate availability Phosphate deprivation Gastrointestinal malabsorption Transcellular shift of phosphate Alkalosis Glucose administration

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Disorders of Phosphate Homeostasisdcont’d

Combined mechanisms Alcoholism Burns Nutritional recovery syndrome Diabetic ketoacidosis Hyperphosphatemic syndromes Vitamin D intoxication Rhabdomyolysis Cytotoxic therapy Malignant hyperthermia

dominant hypophosphatemic rickets (ADHR). These phosphatonin(s) regulate renal phosphate transport and bone mineralization and participate in the pathophysiological cascade of events underlying many of the renal phosphate wasting disorders. In fact, current theories have emerged, which suggest that a common metabolic pathway underlies many of these hypophosphatemic diseases. The Common Pathway Underlying the Pathogenesis of TIO, ADHR, ARHR, and XLH As detailed below, XLH, ADHR, and ARHR are genetic disorders characterized by hypophosphatemia, due to impaired renal tubular reabsorption of phosphate, inappropriately normal or decreased serum 1,25(OH)2D levels, and defective cartilage and bone mineralization. In contrast TIO is an acquired hypophosphatemic disorder caused by production of a factor(s) by tumors, which leads to phenotypic features similar to those of the hereditary phosphate wasting diseases. Based on the shared phenotype of these disorders, several groups have postulated the existence of a unique circulating protein(s), common to each of these diseases, which inhibits sodiumdependent phosphate reabsorption by the renal proximal tubule through PTH distinct mechanisms, impairs bone and cartilage mineralization, and deters hypophosphatemia-mediated increments in the renal production of 1,25(OH)2D. Initial evidence lending credence to the possibility that there is such a common metabolic pathway underlying these diseases derived from studies of patients with TIO. By culturing a tumor associated with this disease, Cai et al. [31] demonstrated that supernatants of such tumor cells, maintained in culture, contained a factor(s) that inhibited sodium-dependent phosphate reabsorption in renal cultured epithelia. The effects of this factor(s) were specific, cyclic AMP independent,

and distinct from those of PTH. These data, as well as subsequent reports from other groups [32e34], indicated the existence of a novel substance(s), putatively named phosphatonin(s) [35], that alters phosphate reabsorption in the renal tubule. Further investigation, utilizing serial analysis of gene expression and array profiling, resulted in identification of at least 10 genes consistently overexpressed in tumors from patients with TIO, relative to patients with mesenchymal tumors but without TIO [36]. These overexpressed genes included those that encode fibroblast growth factor (FGF) 23, secreted frizzle-related protein (sFRP)-4, matrix extracellular phosphoglycoprotein (MEPE), dentine matrix protein 1 (DMP1), and PHEX [37e41]. Although, in the vast majority of patients with surgically confirmed TIO, hormone overproduction by the tumor results in secretion of FGF23, as evidenced by the elevated serum concentration of this hormone in 92% of such patients [42], the independent role of this phosphatonin in the genesis of the syndrome has not been established. The possibility that these observations linked the pathogenesis of TIO to that of ADHR was suggested by the seminal discovery that FGF23 has a pathogenetic role in patients with ADHR [43,44]. Patients with this syndrome have missense mutations in the FGF23 gene at the subtilisin-like proprotein convertase (SPC) cleavage site, 176-RXXR-179 (R176Q, R179W, and R179Q), rendering the FGF23 protein resistant to cleavage/hydrolysis and inactivation by SPC proteolytic enzymes [43,45]. The resultant increase in circulating levels of cleavage-resistant FGF23 was presumed responsible for phosphaturia and abnormal bone mineralization due to direct actions of this hormone on the renal proximal tubule and the osteoblasts. Since tumors in patients with TIO overexpress and hypersecrete FGF23 and mutations in the FGF23 gene underlie ADHR, the possibility that FGF23 is the presumed phosphatonin operative in these diseases seemed plausible. Subsequent studies established the biological activity of FGF23, an approximately 26 kDA circulating protein, consisting of an N-terminal FGF homology domain and a novel 71-amino-acid C-terminus of uncertain function. Shimada et al. [40] reported that administration of biosynthetically prepared full-length FGF23 to mice resulted in phosphaturia, but did not lead to upregulation of 1,25(OH)2D production. Bai et al. [46] also discovered that intact FGF23 has enhanced in vivo biological potency and tumors derived from cells that overexpressed FGF23 caused a more severe osteomalacia and rickets in nude mice than observed due to hypophosphatemia alone, suggesting a direct effect of FGF23 on cartilage and bone. In concert, Bowe et al. [38] found that FGF23 inhibited sodium-dependent phosphate

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transport in cultured renal epithelia by decreasing the expression of the NPT2a mRNA and protein. Conversely, FGF23-null mice exhibited elevated serum phosphorus levels and increased 1,25(OH)2D production, confirming an essential and non-redundant role of FGF23 in regulation of phosphate homeostasis [47], while FGF23 transgenic mice exhibited a phenotype consistent with a severe form of ADHR [48]. In addition, several groups have found that circulating FGF23 levels are elevated in patients with ADHR, who manifest hypophosphatemia and other elements of the disorder, as in patients with TIO [49e51]. Moreover, further studies have documented that the well-known variability of symptoms and disease severity in patients with ADHR fluctuate with the serum FGF23 concentration [52]. Absent from these studies, however, is documentation that FGF23 has an independent role in the genesis of the rickets/osteomalacia characteristic of ADHR or if the rickets/osteomalacia in patients with ADHR is phosphate-responsive or -resistant. More recent studies have determined that the phenotype of autosomal recessive hypophosphatemic rickets (ARHR) bears a striking resemblance to that of both TIO and ADHR. ARHR, characterized by renal phosphate wasting, aberrant regulation of 25(OH)D-1ahydroxylase activity and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). A relationship between ARHR, TIO, and ADHR was assured by documentation of elevated circulating levels of FGF23 in patients with ARHR and its Dmp1-null mouse homolog [53e56]. The common pathophysiological basis for these diseases was thereafter confirmed by the observation that Dmp1 deficiency in the mouse homolog of ARHR also results in a selective increase in osteocyte production of FGF23. In addition, the recognition that ablation of Fgf23 in Dmp1-null mice results in increased serum phosphorus and 1,25(OH)2D levels in the Dmp1-null mice indicates an independent role for FGF23 in regulation of phosphate and 1,25(OH)2D levels in ARHR. These observations provide unprecedented evidence that links the pathophysiological cascade underlying TIO, ADHR, and ARHR. Nevertheless, the absence of studies addressing the mechanisms whereby Dmp1 deficiency stimulates transcription of FGF23 in osteocytes and inhibits bone mineralization preclude determining that FGF23 alone underlies the bone phenotype (osteomalacia/rickets) in this disorder. Indeed, hypophosphatemia and/or the presence of an alternative factor (similar to that possibly present in TIO) may complement the effects of FGF23 in the genesis of the ARHR phenotype. However, the recent observation that the ARHR phenotype is rescued by overexpression of the Dmp1 57 kDa C-terminal fragment either supports that FGF23 alone underlies the phenotype or suggests that

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Dmp1 deficiency alters another factor(s) that regulates bone mineralization [57]. Studies of XLH have further reinforced that FGF23 is the phosphatonin, providing a common link to the pathogenesis of the phosphate wasting diseases and provided insight to the enigma surrounding the role that FGF23 plays in regulation of bone and cartilage mineralization. Early studies by Meyer et al. [58], testing parabiosis between Hyp and normal mice, suggested the presence of a circulating factor that causes hypophosphatemia in the mouse model of XLH. In concert, Lajeunesse et al. [59] discovered the presence of a serum factor in Hyp mice that inhibits phosphate transport in renal epithelia, and others reported that a similar factor is elaborated by Hyp mouse osteoblasts, which not only inhibits renal phosphate transport but impairs osteoblast mineralization [60,61]. Further, renal cross-transplantation studies confirmed the presence of a phosphate transport inhibitory factor in Hyp mice and excluded the possibility that XLH is due to an intrinsic defect in the renal phosphate co-transport system [62]. Upon discovery that mutations of the PHEX gene underlie XLH (see below), a series of observations established the interrelated events linking the HYP phenotype to FGF23. Most notably, the absence of Phex in the kidneys of Hyp mice [63e69] indicated that the gene mutation must indirectly regulate the expression of NPT2a/Npt2a in renal tubular cells. In accord, several groups [70e72] documented that inactivation of Phex in the osteoblasts or osteocytes of Hyp mice leads to decreased Fgf23 proteolysis and to upregulation of Fgf23 mRNA expression, which collectively increase circulating Fgf23 levels not only in Hyp mice [72], but in the vast majority of patients with XLH [49,50]. Interestingly, in Hyp mice and patients with XLH, the mechanisms underlying the elevated circulating FGF23 levels, limited FGF23 degradation and increased FGF23 mRNA production, are the same that occur in patients with ADHR and TIO/ARHR, respectively. The elevated FGF23 levels in XLH promote phosphaturia, and subsequent renal phosphate wasting may contribute to unmineralized cartilage/bone, as noted in both Hyp mice and XLH. The observed amelioration of hypophosphatemia and rickets in Hyp mice, secondary to antibody-mediated neutralization of FGF23, supports this role for FGF23 [72]. Information regarding the downstream effects by which inactivating mutations of PHEX decrease FGF23 proteolysis and enhance FGF23 mRNA production, thereby increasing serum FGF23 levels, remains incomplete. Indeed, despite extensive characterization of the PHEX/Phex gene and PHEX/Phex protein in humans and mice, repeated studies over the past 10 years have

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63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS

failed to definitively identify a substrate for the protein [38,73e80] or the downstream effects of PHEX/Phex, which result in the classical renal and bone phenotype in XLH [48,76,81,82]. In this regard, several studies have definitively excluded FGF23/Fgf23 as a substrate for PHEX/Phex [38,73,74]. Moreover, investigations to establish that changes in serum P mediate alterations in serum FGF23/Fgf23, and thereby hormone degradation or production, are variable and inconclusive [83e87]. Thus, the ability to alter FGF-23 and positively influence human diseases, through modification of PHEX interaction with FGF-23 or an alternative substrate, has not been possible. More recently, however, the downstream Phex-dependent effects that modulate Fgf-23 production and degradation have been elucidated. These studies revealed not only that Fgf-23 degradation and production occur primarily in the osteoblast/osteocyte but are seemingly regulated by 7B2 and 7B2•SPC2 activity, a subtilisin-like proprotein convertase. In these investigations Yuan et al. (88) documented that a decrease in the 7B2 chaperone protein mRNA in Hyp mouse osteoblasts, and consequent diminished 7B2•SPC2 enzyme activity, limits Fgf23 degradation and enhances Fgf23 mRNA production. Moreover, the effects on Fgf23 production are mediated by a downstream effect of decreased 7B2•SPC2 enzyme activity, impaired Dmp1 degradation and resultant deficiency of the 57 kDa C-terminal proteolytic Dmp1 fragment, which is a mechanism identical to that by which FGF23 is increased in ARHR (Fig. 63.3). Collectively, the aforementioned observations form the basis of the model for a common pathogenesis of XLH, TIO, ADHR, and ARHR. According to this model, FGF23 is the phosphatonin that inhibits sodium-dependent phosphate uptake in the renal proximal tubule and contributes, by an unknown mechanism, to impaired bone mineralization. This model presumes that only fulllength FGF23 is phosphaturic and impacts mineralization. Further, the model predicts that FGF23 is increased: (1) in ADHR because mutations in FGF23 render it resistant to subtilisin-dependent proprotein convertasedependent cleavage; (2) in TIO and ARHR because overproduction of FGF23 overwhelms the inactivation capacity of degradative mechanisms; and (3) in XLH because downstream effects of PHEX limit FGF23 degradation and stimulate FGF23 mRNA production. Consistent with this model, multiple studies have linked the biological activities of FGF23 to the phenotype of the hypophosphatemic diseases. However, presumed restoration of normal FGF23 activity in Hyp mice by a variety of techniques fails to normalize renal phosphate transport and hypophosphatemia [72,89e91], albeit flawed experimental design and subsequent data from studies evaluating mice with knockout of the Phex gene negate the message of these investigations.

Nevertheless, there are a number of additional inconsistencies and unexplained observations that raise concern about whether this simple FGF23 hypothesis is correct. First and foremost, the existence of other phosphatonins raises the plausible possibility that proteins other than FGF23 may be operative in TIO, ARHR, and XLH. Second, the known biologic activities of FGF23 do not explain the panorama of phenotypic abnormalities common to the hypophosphatemic disease. Third, the downstream effects of PHEX that regulate FGF23 degradation and production are not unequivocally established. These issues are considered in the remainder of this section. The most compelling of the observations, challenging the FGF23 hypothesis, is the existence of additional phosphatonins. Thus, while it is tempting to speculate that TIO is due solely to excessive production of FGF23, this is not necessarily the case since tumors overexpress the mRNA of other molecules, including sFRP-4 and MEPE [41], that exhibit the characteristics of a phosphatonin. In this regard, Berndt et al. [92] provided compelling evidence that sFRP-4 has unmistakable characteristics of a phosphatonin. Thus, recombinant sFRP-4 inhibits sodium-dependent phosphate transport in cultured opossum renal epithelial cells, indicating a possible direct action of sFRP-4 on proximal tubular phosphate transport. Further, the systemic administration of recombinant sFRP-4 caused phosphaturia (and hypophosphatemia) in normal rats without stimulating renal 25-hydroxyvitamin D-1a-hydroxylase activity. Moreover, the effects on renal epithelia occurred through a mechanism that involved antagonism of Wnt-dependent b-catenin pathways. And, finally, Berndt et al. [92] extended these observations by showing that sFRP-4 is present in normal human serum and in the serum of patients with TIO. Similar experiments have established MEPE as a phosphatonin. In this regard, extensive experiments have documented that MEPE is exclusively expressed in osteoblasts and osteocytes in rodents [89,93e97] and in abundance in human bone, as well as in human brain, albeit in lesser amounts [93,95e97]. Moreover, MEPE expression occurs in all tumors from patients with TIO and is notably absent in non-phosphaturic tumors [37,40,93]. Further, Rowe et al. [98] have demonstrated that MEPE inhibits phosphate transport in renal epithelia in vitro and when administered in vivo to rats results in significant dose-dependent phosphaturia and hypophosphatemia. However, a report regarding the MEPE null-mutant mouse phenotype showed that the MEPE-deficient mice did not have abnormalities of serum phosphorus, as might be expected if MEPE played an important role in phosphate homeostasis [97]. Moreover, Liu et al. [99] reported that transfer of MEPE deficiency onto the Hyp mouse background failed

VIII. DISORDERS

1163

DISORDERS OF PHOSPHATE HOMEOSTASIS

(A)

PHEX/Phex

7B2 mRNA

2 DMP1 (104kd)

ProBMP1

Normal Osteoblasts

SPC2•7B2

SPC2 mRNA

BMP1 DMP1 + DMP1 (57kd) (37kd)

1 Normal Degradation

C-term Fgf23 + N-term Fgf23

Fgf23

Suppressed Production

Fgf-23 mRNA SOST mRNA

(B) PHEX/Phex Hyp Osteoblasts

7B2 mRNA ProBMP1

SPC2 mRNA

SPC2•7B2

DMP1 (104kd)

BMP1 Decreased Degradation Increased Fgf-23

Fgf23

C-term Fgf23 + N-term Fgf23

Decreased Increased Production

Increased

DMP1 + (57kd)

DMP1 (37kd)

Fgf-23 mRNA SOST mRNA

FIGURE 63.3

(A) In the normal osteoblast a series of reactions occur that regulate the degradation of Fgf23 (box 1) and the production of Fgf23 and Sclerostin (box 2). In ADHR a genetic mutation of Fgf-23 alters the cleavage site, precluding normal degradation of the full-length bioactive protein and resulting in an accumulation of the Ffg23. The attendant increased serum Fgf23 concentration results in the characteristic biochemical phenotype in ADHR. In ARHR an inactivating mutation of DMP1 limits the production of the 57kd C-terminal fragment of DMP1, which normally suppresses production of Fgf23 and SOST mRNA and thereby the coded proteins Fgf23 and Sclerostin. The resultant increased serum Fgf23 concentration causes renal phosphate wasting and the characteristic biochemical phenotype, whereas the increased serum Fgf23 and Sclerostin are apparently responsible for the bone mineralization abnormality in ARHR. (B) In the osteoblast from the Hyp-mouse, an inactive mutation of PHEX/Phex causes a downstream effect, decreased 7B2 mRNA. The resultant decreased 7B2 protein decreases the SPC2•7B2 enzyme activity. The diminished enzyme activity retards Fgf23 degradation and increases the concentration of full-length bioactive Ffg23. The decreased enzyme activity also diminishes conversion of ProBMP1 to BMP1, which limits the degradation of DMP1 to its 57kd Cterminal fragment. The decreased carboxy-terminal fragment lifts suppression of Fgf23 and SOST mRNA, resulting in increased transcription and respective protein concentrations. The increased Fgf23 causes renal phosphate wasting and the characteristic biochemical phenotype, whereas the increased serum Fgf23 and Sclerostin are apparently responsible for the bone mineralization abnormality in XLH. Thus, in XLH abnormalities of the processes in box 1 and box 2 (A) occur. The box 1 abnormality, in essence, recapitulates the nature of the abnormality in ADHR, while that in box 2 is similar to that in ARHR. It is important to note that in other renal phosphate wasting diseases in humans, most notably TIO, the pathophysiology simply involves increased production of Fgf23 and possibly Sclerostin, giving rise to a similar phenotype but not requiring the genetic mutations that result in the increased mRNA production.

to rescue the hypophosphatemia in the mutants, suggesting that MEPE may not cause phosphaturia in the setting of inactivating Phex mutations. Thus, it is possible that MEPE may influence only bone mineralization and not phosphate homeostasis, thereby requiring the tumor secretion of a complementary phosphatonin to create the complete TIO phenotype. The potential effects of MEPE on bone mineralization have been supported in ancillary studies. First, MEPE-deficient mice exhibit increased bone formation and mineralization, consistent with the possibility that MEPE, under physiological conditions, plays an inhibitory role in bone mineralization and formation. Second, Rowe et al. [98] documented that MEPE dose-dependently inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Thus, the ever-present MEPE

overproduction in tumors of patients with TIO precludes establishing with certainty an independent role of FGF23 in genesis of the syndrome. Similar uncertainty about the independent role of FGF23 in the genesis of the HYP phenotype exists. Indeed, multiple studies have identified that the production rate and/or the circulating level of the phosphatonins, FGF23, MEPE, and sFRP-4, are increased in patients with XLH and/or Hyp mice [100]. However, Yuan et al. [100] found that while Hyp mice and mice with global knockout of Phex (Cre-PhexDflox/y) exhibited increased production and serum levels of FGF-23, MEPE, and sFRP-4, mice with a targeted knockout of Phex in osteoblasts (OC-Cre- PhexDflox/y), despite manifesting the characteristic HYP phenotype, had only an increased osseous production rate and serum level of

VIII. DISORDERS

1164 TABLE 63.2

63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS

Activity of Candidate Phosphatonin/Minhibin Proteins FGF23

sFRP4

MEPE

In vitro

?

?

þ

In vivo

þ

?

?

In vitro

þ

þ

þ

In vivo

þ

þ

þ

In vitro

þ

þ

e

In vivo

þ

þ

e

In vitro

Z

e

e

In vivo

Z

e

\

In vitro

?

?

?

In vivo

?

?

?

Bone Inhibits mineralization

Kidney Inhibits Naþ-Pi transport

Decreases Npt2a transcription

Vitamin D metabolism Alters 25(OH)D-1a-hydroxylase mRNA

Alters 25(OH)D-1a-hydroxylase protein

Fgf23. These data suggest that increased bone production and serum levels of MEPE and sFRP-4 are not critical for development of the classical HYP phenotype, whereas increased osseous production and serum Fgf23 concentration appear requisite for this biological function. Indeed, the observations that a combined Fgf23-deficient and Phex-deficient mouse model displayed no evidence of abnormal phosphate homeostasis supports the conclusion that FGF23 is the sole phosphatonin that contributes to the genesis of the HYP phenotype [71]. However, these data do not establish that FGF23 is solely responsible for the bone mineralization abnormality in these diseases. Indeed, available studies do not preclude that a non-phosphatonin protein may complement the effects of FGF23 to create the bone phenotype in the hypophosphatemic diseases. Alternatively, in some of the hypophosphatemic diseases the bone abnormality may be only a mild form of phosphate-dependent rickets/osteomalacia. The inadequate evidence supporting FGF23 as the inhibitor of bone mineralization is best summarized in Table 63.2. As illustrated, studies do not yet exist that demonstrate the in vitro inhibitory activity of FGF23 on osteoblast mineralization. Yet, studies in Hyp mice

and/or transgenic Fgf23 mice clearly indicate that the mineralization defect in the hypophosphatemic disorders exceeds that expected from hypophosphatemia alone and does not normalize with phosphate therapy. Hence, an effect of FGF23 on osteoblast mineralization function superficially seems certain. However, Yuan et al. [101] have presented data from comparative studies of Hyp mice and mice with targeted knockout of Phex in osteoblasts (OC-Cre- PhexDflox/y) and osteocytes (DMP1-Cre-PhexDflox/y), which challenge a singular role for FGF23 in the regulation of bone mineralization. Despite an identical biochemical phenotype of hypophosphatemia, renal phosphate wasting, and abnormal vitamin D metabolism, and an equally elevated serum Fgf23 level in each of these murine models, only the Hyp mice and those with a targeted knockout of Phex in osteoblasts had severe rickets and osteomalacia, which did not ameliorate in response to phosphate loading. In contrast, mice with targeted knockout of Phex in osteocytes had a very mild mineralization disorder, which was significantly different from that in the Hyp and OC-Cre- PhexDflox/y mice and, in contrast, completely resolved following phosphate loading. These observations challenge the assumption that increased circulating levels of FGF23 underlie the abnormal bone and cartilage mineralization in XLH. Rather, the data suggest that a factor(s) downstream of Phex, other than FGF23 and the serum phosphorus concentration, is variably controlled by the inactivating PHEX mutation and plays a pivotal role in the regulation of bone mineralization in XLH. Preliminary studies by Yuan et al. [101] indicate that elevated Sclerostin, which is present in Hyp and OC-Cre-PhexDflox/y mice, but not in DMP1-Cre-PhexDflox/y mice, may serve as the pivotal factor underlying the abnormal bone mineralization in XLH. This possibility is supported by recent investigations that document increased Sclerostin is likewise present in the Dmp1-null mouse, the murine model for ARHR. Moreover, the 57 kDa C-terminal rescue of the Dmp1-null mouse phenotype is associated with normalization of the elevated Sclerostin, consistent with the role that this protein may play in abnormal bone mineralization (Fig. 63.3). Further, although De Beur et al. [41] only identified FGF23 and sFRP-4 as the candidate genes for the proteins underlying TIO, because only 67 of the original 364 candidate genes identified by SAGE from the tumors evaluated were validated by array analysis or RT-PCR, it is plausible that the SOST gene, encoding Sclerostin, is overexpressed in TIO and possibly the cause of abnormal mineralization in this syndrome. Interestingly, the anticipated effects of phosphatonins (most notably FGF23) on vitamin D metabolism are also controversial. Although phosphate-mediated enhancement of calcitriol production and serum levels is

VIII. DISORDERS

DISORDERS OF PHOSPHATE HOMEOSTASIS

uniformly inhibited in ADHR, TIO, ARHR, and XLH, the cause of this abnormality in each disorder is less certain. Since regulation of calcitriol production occurs primarily at the transcription level, several groups have anticipated that a phosphatonin would inhibit renal 1a-hydroxylase transcription. In accord, current studies indicate that FGF23 fulfills this criterion both in vitro and in vivo (Table 63.2). However, investigations in the Hyp mouse indicate that renal 1a-hydroxylase mRNA is, in fact, elevated under basal conditions and following PTH stimulation [102]. Therefore, the inhibition of calcitriol production occurs at the translational level [103]. As shown in Table 63.2, available data have not linked any of the phosphatonins to this phenotypic characteristic of XLH. Confounding this issue further, Yuan et al. [104] recently reported that the effects of FGF23 on 1a-hydroxylase activity are not direct but are mediated by abnormal renal phosphate transport. Thus, Hyp mice, with transgenic overexpression of Npt2a mRNA and consequent normal serum phosphorus levels, do not exhibit aberrant regulation of 1,25(OH)2D production. These observations suggest that the effects of FGF23 on vitamin D metabolism are mediated by a hormone-induced decrease in renal phosphate transport, which results in abnormal translational regulation, likely due to inadequate P450 phosphorylation and consequent ineffective bimodal targeting of 1a-hydroxylase protein to mitochondria, the site of enzyme activity [105e110]. These remarkable advances in understanding the abnormal regulation of vitamin D metabolism in Hyp mice and likely patients with XLH do not explain the discrepant observations regarding FGF23 effects on 1,25(OH)2D production in vitro and in vivo, which are mediated by decreased 25(OH)D-1a-hydroxylase gene transcription. Thus, further studies are essential in order to determine which elements of the HYP phenotypic milieu alter the effects of FGF23 on vitamin D metabolism and ascertain if regulation of 1,25(OH)2D production in other hypophosphatemic disorders is similar to that in XLH. Over the past decade, the progress made in defining a common pathogenetic mechanism for these hypophosphatemic diseases has been truly remarkable and enhanced our understanding of the various disorders of phosphate homeostasis. Indeed, based on these advances, many investigations are under way to create new treatment strategies for these hypophosphatemic disorders. Moreover, further progress in the next decade will undoubtedly resolve the details of the common FGF23-dependent pathogenetic mechanism underlying ADHR, TIO, ARHR, and XLH. Consideration of these diseases in the remainder of this chapter will highlight many of these advances. Chapter 26 and Chapter 42 have further discussion of the phosphatonins.

1165

X-linked Hypophosphatemia (XLH) XLH is the prototypic renal phosphate wasting disorder, characterized by progressively severe skeletal abnormalities and growth retardation. The syndrome occurs as an X-linked dominant disorder with complete penetrance of a renal tubular abnormality resulting in phosphate wasting and consequent hypophosphatemia (Table 63.3). The clinical expression of XLH varies widely, ranging from a mild presentation with apparently isolated hypophosphatemia, to severe rickets and/or osteomalacia [111]. In children, the most common clinically evident manifestations include short stature and limb deformities. This height deficiency is more evident in the lower extremities, since they represent the fastest-growing segment before puberty. In contrast, upper segment growth is generally less affected. The majority of children with the disease exhibit enlargement of the wrists and/or knees secondary to rickets, as well as bowing of the lower extremities. Additional signs of the disease may include late dentition, tooth abscesses secondary to poor mineralization of the interglobular dentine, and premature cranial synostosis. Many of these features are not apparent until 6 to 12 months of age or older [112]. Despite marked variability in the clinical presentation, bone biopsies in affected children and adults invariably reveal low turnover osteomalacia without osteopenia. The severity of the bone disorder has no relationship to gender, the extent of the biochemical abnormalities, or the degree of the clinical disability. In untreated youths and adults, the serum 25(OH)D levels are normal and the concentration of 1,25(OH)2D is in the lowenormal range [113e115]. The paradoxical occurrence of hypophosphatemia and normal serum calcitriol levels is due to aberrant regulation of renal 25(OH)D1a-hydroxylase activity. Studies in Hyp and Gy mice, the murine homologs of the human disease, have established that defective regulation is confined to the enzyme localized in the proximal convoluted tubule, the site of the abnormal phosphate transport [116e119]. PATHOPHYSIOLOGY

Investigators generally agree that the primary inborn error in XLH results in an expressed abnormality of the renal proximal tubule that impairs Pi reabsorption. This defect has been indirectly identified in affected patients and directly demonstrated in the brush border membranes of the proximal nephron in Hyp mice. Whether this renal abnormality is primary or secondary to the elaboration of a humoral factor was controversial. In this regard, demonstration that renal tubule cells from Hyp mice maintained in primary culture exhibit a persistent defect in renal Pi transport [120,121], likely due to decreased expression of NPT2a mRNA and

VIII. DISORDERS

1166 TABLE 63.3

63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS

Biochemical/genetic characteristics of the prototypic phosphopenic disorders in humans FS XLH

TIO

ADHR

ARHR

HHRH

I

II

TC

Serum P

Z

Z

Z

Z

Z

Z

Z

\

Renal TmP/GFR

Z

Z

Z

Z

Z

Z

Z

\

GI P absorption

Z

Z

Z

Z

\

Z

\

\

FGF-23

N or \

\

\

\

?

?

?

?

Serum Ca

N

N

N

N

N

N

N

N

Urine Ca

Z

Z

Z

Z

\

Z

\

\

Nephrolithiasis

e

e

e

þ

e

e

e

e

GI Ca absorption

Z

Z

Z

Z

\

Z

\

\

Serum PTH

N

N

N

N

N

N

N

N

P HOMEOSTASIS

Ca HOMEOSTASIS

VITAMIN D METABOLISM 25(OH)D

N

N

N

N

N

N

N

N

1,25(OH)2D

N/Z

Z

N/Z

Z

\

N/Z

\

\

Serum Alk Phos

N/\

N/\

N/\

N/\

N/\

N/\

N/\

N

Serum NPT

N

N

N

N

N

N

N

?

Familial

þ

e

þ

þ

þ

Variable

þ

þ

Transmission

X-linked dominant

e

Autosomal dominant

Autosomal recessive

Autosomal recessive

Variable

?

Variable

Abnormal gene

PHEX

e

FGF-23

DMP1

NPT2c

Variable

?

?

BONE METABOLISM

GENETICS

XLH, X-linked hypophosphatemia; TIO, tumor-induced osteomalacia; HHRH, hereditary hypophosphatemic rickets with hypercalciuria; FS I, Fanconi syndrome, type I; FS II, Fanconi syndrome, type II; ADHR, autosomal dominant hypophosphatemic rickets; ARHR, autosomal recessive hypophosphatemic rickets; TC, tumoral calcinosis. N, normal; Z, decreased; \, increased. Modified from [237].

immunoreactive protein [122e124], supported the presence of a primary renal abnormality. In contrast, transfer of the defect in renal Pi transport to normal and/or parathyroidectomized normal mice, parabiosed to Hyp mice, implicated a humoral factor in the pathogenesis of the disease [58,125]. Current studies, however, have provided compelling evidence that the defect in renal Pi transport in XLH is secondary to the effects of a circulating hormone or metabolic factor. In this regard, immortalized cell cultures from the renal tubules of Hyp and Gy mice exhibit normal Naþ-phosphate transport, suggesting that the paradoxical effects observed in primary cultures may represent the effects of impressed memory and not an intrinsic abnormality [126,127]. Moreover, the report that cross-transplantation of kidneys in normal and Hyp mice results in neither transfer of the mutant phenotype or its correction

unequivocally established the humoral basis for XLH [62]. Subsequent efforts, which resulted in localization of the gene encoding the Naþ-phosphate co-transporter to chromosome 5, further substantiated the conclusion that the renal defect in brush border membrane phosphate transport is not intrinsic to the kidney in XLH [128]. These data established the presence of a humoral abnormality in XLH. Subsequently, several investigators identified and characterized the biological activities of a variety of phosphaturic factors (inhibitors of Naþdependent phosphate transport) and mineralization inhibitory factors, which may play a role in the pathogenesis of XLH (see above). Moreover, several reports have documented production of phosphaturic and mineralization inhibitory factors by Hyp mouse osteoblasts maintained in culture [61,89,93,126,129]. Therefore, as noted above, these studies argue that

VIII. DISORDERS

DISORDERS OF PHOSPHATE HOMEOSTASIS

a circulating factor(s), phosphatonin(s), FGF23, plays an important role in the pathophysiological cascade responsible for X-linked hypophosphatemia. GENETIC DEFECT

Efforts to better understand XLH have led to identification of the genetic defect underlying this disease. In 1986 Read et al. [130] and Machler et al. [131] reported linkage of the DNA probes DXS41 and DXS43, which had been previously mapped to Xp22.31-p21.3, the HYP gene locus. In subsequent studies Thakker et al. [132,133] reported linkage to the HYP locus of additional polymorphic DNA, DXS197, and DXS207 and, using multipoint mapping techniques, determined the most likely order of the markers as Xpter-DXS85-(DXS43/ DXS197)-HYP-DXS41-Xcen and Xpter-DXS43-HYP(DXS207/DXS41)-Xcen, respectively. The relatively small number of informative pedigrees available for these studies prevented definitive determination of the genetic map along the Xp22-p21 region of the X-chromosome and only allowed identification of flanking markers for the HYP locus 20 centimorgans (cM) apart. However, the independent and collaborative efforts of the HYP consortium resulted in the study of 13 multigenerational pedigrees and consequent refined mapping of the Xp22.1-p21 region of the X chromosome, identification of tightly linked flanking markers for the HYP locus, construction of a YAC contig spanning the HYP gene region, and eventual cloning and identification of the disease gene as PHEX, a Phosphate regulating gene with Homologies to Endopeptidases located on the X-chromosome. In brief, these studies ascertained a locus order on Xp22.1 of: Xcen  DXS451  ðDXS41=DXS92Þ  DXS274  DXS1052  DXS1683  PHYP  DXS7474  DXS365  ðDXS443=DXS3424Þ  DXS257  ðGLR=DXS43Þ  DXS3 15  Xtel Moreover, the physical distance between the flanking markers, DXS1683 and DXS7474, was determined as 350 kb and their location on a single YAC ascertained. Subsequently, a cosmid contig spanning the HYP gene region was constructed and efforts directed at discovery of deletions within the HYP region. Identification of several such deletions permitted characterization of cDNA clones that mapped to cosmid fragments in the vicinity of the deletions. Database searches with these cDNAs detected homologies at the peptide level to a family of endopeptidase. These efforts clearly established PHEX as the candidate gene responsible for XLH [134e139]. Identification of the gene associated with XLH as PHEX [134] has facilitated efforts to better understand

1167

this disease. The gene codes for a 749-amino-acid protein, consisting of three domains: (1) a small aminoterminal intracellular tail; (2) a single, short transmembrane peptide; and (3) a large carboxyterminal extracellular peptide, which, typical of zinc metalloproteases [140], has 10 conserved cysteine residues and a HEXXH pentapeptide motif. The homology of PHEX with metalloproteases resulted in inclusion of this protein in the M13 family of membrane-bound metalloproteases, also known as neutral endopeptidases [141e143]. M13 family members, including neutral endopeptidase 24.11 (NEP), endothelin-converting enzymes 1 and 2, the Kell blood group antigen, neprilysin-like peptide (NL1), and endothelin converting enzyme-like 1 [140,142,144e150], degrade or activate a variety of peptide hormones. Preservation in the PHEX structure of catalytic glutamate and histidine residues (equivalent to Glu648 and His711 of NL1) argues for similar protease activity, as does alignment of PHEX mutations with regions required for peptidase activity in NL1 [151]. Further, like other neutral endopeptidases, immunofluorescent studies reveal a cell-surface location for PHEX in an orientation consistent with a type II integral membrane glycoprotein [151]. In any case, cloning the PHEX gene led relatively rapidly to cloning the homologous murine Phex gene and identification of the mutations in the murine homologs of XLH, the Hyp and Gy mice [64,152,153]. Unlike 97% of known genes, neither the human nor murine gene has a Kozak sequence, a purine at the e3 position before the ATG initiation sequence [152e154]. Since such genes are often posttranscriptionally regulated, this anomaly may impact understanding the hormonal and metabolic regulation of PHEX/Phex. Many investigators [63e69,134,154e162] have used Phex/Phex localization and mutation detection to help formulate the pathogenetic scheme for XLH. Investigation of murine tissues and cell cultures revealed that Phex is predominantly expressed in bones and teeth [63,64,66e68,153], while mRNA, protein or both have also been found in lung, brain, muscle, gonads, skin, and parathyroid glands [151,163]. Experiments in neonatal and adult mice further documented that the cellular locations of Phex in bone and teeth are the osteoblast/osteocyte and the odontoblast/ameloblast, respectively, while subcellular locations are the cell-surface membrane, the endoplasmic reticulum, and the Golgi compartment. Notably Phex expression is absent in the visceral abdominal organs, including the kidney, liver hepatocytes, and intestine, and in cardiac and skeletal muscle. In any case, the ontogeny of Phex expression reveals that the protein is expressed in osteoblasts at both primary and secondary ossification centers, suggesting a possible role in mineralization in vivo.

VIII. DISORDERS

1168

63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS

To date >200 different PHEX mutations, consisting of deletions, frameshifts, insertions, and duplications, as well as splice site, frameshift, nonsense, and missense mutations, have been documented in >250 patients with XLH [134,154e161,164] and are scattered throughout exons 2e22, which encode the 749-amino-acid extracellular protein domain. In addition, a single mutation within the 50 untranslated region has been identified [157]. Although these mutations invariably cause loss of function, the mechanism by which such loss of activity occurs is unclear. However, preliminary data indicate that missense mutations interfere with protein trafficking, resulting in protein sequestration in the endoplasmic reticulum [160]. Since PHEX coding region mutations had not been detected in ~35% of patients, Christie et al. [161] explored whether such subjects have intronic mutations that result in mRNA splicing abnormalities. They found in one patient a unique mutation in intron 7 that created a novel donor splice site, which interacts with three naturally occurring acceptor splice sites, leading to the incorporation of three pseudoexons in PHEX transcripts. Translation of these pseudoexons results in the inclusion of missense amino acids into the PHEX protein or a truncated protein, lacking five of ten conserved cysteine residues and the pentapeptide zinc-binding motif. These observations suggest that intron mutations may represent a proportion of the gene abnormalities undiscovered in one-third of patients with XLH. In order to confirm the possibility that diminished PHEX/Phex expression in osteoblasts initiates the cascade of events responsible for the pathogenesis of XLH, several investigators have used targeted overexpression of Phex in attempts to normalize osteoblast mineralization, in vitro, and rescue the Hyp phenotype in vivo. Surprisingly, however, these studies [90,165] revealed that restoration of Phex expression and enzymatic activity to immortalized Hyp mouse osteoblasts, by retroviral-mediated transduction, does not restore their capacity to mineralize extracellular matrix in vitro, under conditions supporting normal mineralization. Moreover, in complementary studies Liu et al. [90] and Bai et al. [89] found transgenic Hyp mice (OscPhex-Hyp; pOb2.3[ColIaI]-Phex-Hyp), despite expressing abundant Phex mRNA and enzyme activity in mature osteoblasts and osteocytes, exhibited hypophosphatemia and persistently abnormal vitamin D metabolism. In the setting of P depletion, although exhibiting a modest improvement in bone mineralization, the transgenic mice maintained histological evidence of osteomalacia, similar to that in non-transgenic Hyp mice. These observations are consistent with several possibilities, acknowledged by Liu et al. [90] and Bai et al. [89], which suggest experimental design flaws in the study.

First, despite theoretical evidence to the contrary (see above), extraosseous Phex expression may play an important role in the modulation of phosphatonin activity. In support of this option, Miyamura et al. [166] using syngeneic bone marrow transplantation, were able to partly reverse the biochemical abnormalities in Hyp mice with an engraftment that was not restricted to cells of the osteoblast lineage, but included donor cells to alternate tissues, in many of which PHEX transcripts were detected [64e67]. Second and more likely, the temporal and developmental expression of the Osc and pOb2.3 promoter-driven Phex expression may not mimic the endogenous regulation of Phex. In this regard, the transgenic animals may experience PHEX expression later than, or in osteoblast-related cell subpopulations different from those in normal animals. In fact, neither promoter is expressed in the preosteoblast and the osteocalcin promoter appears at least 4 days later than PHEX in normally developing osteoblasts [89,90]. Thus, lack of Phex activity early in osteoblast development (in pre-osteoblasts or preceding osteocalcin expression in osteoblasts) may result in failure to alter an otherwise immutable osteoblast dysfunction, the continued presence of which contributes to the impairment of mineralization. In accord, later expression of PHEX may not rescue the phenotype, as the immutable change is refractory to endopeptidase activity. However, Erben et al. [91] reported that ubiquitous overexpression of Phex under the control of the bactin promoter in two different mice did not completely resolve the bone mineralization defect and failed to alter the abnormal phosphate homeostasis, raising significant further questions regarding the interaction between the PHEX gene defect and phenotypic expression of the disease. Regardless, in no way do these observations exclude a role for inadequate Phex expression in the genesis of XLH or more particularly in the mineralization process in the mature osteoblast. To further explore the role of the osteoblast and osteocyte in the pathogenesis of XLH, Yuan et al. [100] created mice with a global PHEX knockout (Cre-PhexDflox/y mice) and mice with a conditional osteocalcin-promoted PHEX inactivation limited to osteoblasts (OC-Cre-PhexDflox/y mice). Phenotype and bone histology were compared among the two knockout mouse strains, as well as Hyp and normal mice. Elevated Fgf23 levels, reduced brush border membrane phosphate transport, low renal NPT2a protein concentration, and decreased serum phosphorus levels were present in Hyp, Cre-PhexDflox/y and OC-CrePhexDflox/y mice when compared to normal mice. Moreover, Hyp, Cre-PhexDflox/y and OC-Cre-PhexDflox/y mice all demonstrated comparable osteomalacia, providing evidence that abnormal Phex function in osteoblasts alone is sufficient to create the HYP phenotype.

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PATHOGENESIS

In spite of the remarkable advances that have been made in understanding the genetic abnormality and pathophysiology of XLH, the detailed pathogenetic mechanism underlying this disease remains incompletely defined (see above). While as related previously, the Phex substrate remains unknown, a role for FGF23 in the pathogenesis of the disease seems certain. Moreover, it is now clear that a defect in the PHEX gene has downstream effects that result in overproduction and enhanced circulating levels of FGF23 and consequent inhibition of renal Naþ-phosphate transport, the likely operative scenario in the pathogenesis of XLH. The coexistence of osteoblast defects in XLH further confounds understanding the pathophysiology of this disorder. Elegant experiments, which documented the abnormal mineralization of periostea and osteoblasts of Hyp mice following transplantation into the muscle of normal mice, provide clear evidence for intrinsic defects in the bone of mutants [167]. Indeed, proof of specific osteoblast abnormalities has been provided by studies that show decreased phosphorylation of osteopontin and increased osteocalcin levels in cells from Hyp mice [168]. Based on these observations, it is tempting to speculate that FGF23 is not the sole abnormality resulting in abnormal bone mineralization. Indeed, a wide array of studies support this conclusion (see above). Thus, the precise mechanism underlying the abnormal bone mineralization in XLH is not definitively proven. In any case, it is evident that further information is requisite to enhance our understanding of the pathogenesis of XLH and, in turn, regulation of phosphate homeostasis, osteoblast function, vitamin D metabolism, and osteoid mineralization. Such data are not only critical to understanding the pathogenesis of XLH and the regulation of mineral homeostasis, but may have significant impact upon determination of optimal treatment strategies for many of the vitamin-D-resistant diseases. TREATMENT

In past years, physicians employed pharmacologic doses of vitamin D as the cornerstone for treatment of XLH. However, long-term observations indicate that this therapy fails to cure the disease and poses the serious problem of recurrent vitamin D intoxication and renal damage. Therefore, choice of therapy for this disease has been remarkably influenced by the increased understanding of the pathophysiological factors, which affect phenotypic expression of the disorder. Thus, current treatment strategies for children directly address the combined calcitriol and phosphorus deficiency characteristic of the disease. Generally, the regimen includes a period of titration to achieve a maximum dose of calcitriol, 1e3 mg/d in two divided doses and phosphorus,

1e4 g/d in 4e5 divided doses [169,170]. Such combined therapy often improves growth velocity, normalizes lower extremity deformities, and induces healing of the attendant bone disease. Refractoriness to the growth-promoting effects of treatment, however, is often encountered particularly in youths presenting at <5th percentile in height [171]. For that reason, the use of recombinant growth hormone as an additional treatment component has been advocated. Definite positive effects have been observed in young patients with XLH [172,173]. Of course, treatment involves a significant risk of toxicity that is generally expressed as abnormalities of calcium homeostasis, most notably secondary hyperparathyroidism that may become autonomous and require surgery. Detrimental effects on renal function secondary to abnormalities such as nephrocalcinosis are also possible. Thus, frequent monitoring of the phosphate and calcitriol dosage during growth is mandatory. Therapy in adults is reserved for episodes of intractable bone pain and refractory nonunion bone fractures. More recently, addition of calcimimetics as adjuvant therapy in patients with XLH has been advocated [174]. Preliminary observations indicate that inclusion of cinacalcet in the traditional treatment regimen abrogates the anticipated phosphorus-dependent increase in the serum PTH concentration, leading to an increase in the treatment-dependent renal TmP/GFR and serum phosphorus. Such modulation of treatment effects may allow use of lower doses of phosphate and calcitriol. As a consequence the incidence of secondary and tertiary hyperparathyroidism and nephrocalcinosis, known complications of standard therapy, may decrease [175]. Hereditary Hypophosphatemic Hypercalciuria (HHRH)

Rickets

with

This rare genetic disease is marked by hypophosphatemic rickets with hypercalciuria [176]. The cardinal biochemical features of the disorder include hypophosphatemia due to increased renal phosphate clearance and normocalcemia. In contrast to other diseases in which renal phosphate transport is limited, patients with HHRH exhibit increased 1,25(OH)2D production [176e180] (Table 63.3). The resultant elevated serum calcitriol levels enhance the gastrointestinal calcium absorption, which in turn increases the filtered renal calcium load and inhibits parathyroid secretion. These events cause the characteristic hypercalciuria observed in affected patients. The clinical expression of the disease is heterogeneous. In general, children become symptomatic between the ages of 6 months and 7 years. Initial symptoms consist of bone pain or deformities of the lower limbs (or both), which progressively interfere with gait

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and physical activity. The bone deformities vary from genu varum or genu valgum to anterior external bowing of the femur and coxa vara. Additional features at presentation include short stature with disproportionately short lower limbs, muscle weakness, and radiological signs of rickets or osteomalacia (or both). These various symptoms and signs may exist separately or in combination and may be present in a mild or severe form. A large number of apparently unaffected relatives of patients with HHRH exhibit an additional mode of disease expression [180]. These subjects, although without evidence of bone disease, manifest idiopathic hypercalciuria (IH), most evident in postprandial periods, as well as a pattern of biochemical abnormalities similar to those of children with rickets and osteomalacia. Quantitatively, however, the abnormalities are milder, and the relevant biochemical values intermediate between those observed in family members with HHRH and those in normal relatives. The absence of bone disease in these patients may be explained by relatively mild hypophosphatemia compared to the severe phosphate depletion evidenced in patients with the full spectrum of the disorder [180]. Relatively few unrelated kindreds with HHRH have been described, including an extended family of Bedouin origin that includes 13 patients with HHRH and 42 with hypercalciuria; a smaller kindred of oriental Jewish origin with five affected members; and a family of Yemenite Jewish origin that includes two patients with HHRH and two with hypercalciuria [178]. However, several small families with similar genetic abnormalities as those established in patients with classical HHRH have presented with phenotypic variants of the HHRH disease. The variations include: (1) presentation with hypercalciuria and nephrocalcinosis, as well as rickets/osteomalacia, but an associated normal serum calcium and phosphorus and elevated serum calcitriol concentration [181] and (2) a phenotypically similar disorder, including mild childhood idiopathic hypercalciuria with bone lesions (rickets) and stunted linear growth, which is associated with a heterozygotic mutation and variable penetrance [182]. Moreover, several patients with a sporadic occurrence of HHRH have been recognized. Studies are generally incomplete, however, and the presence of hypercalciuria in relatives has not been excluded. PATHOPHYSIOLOGY

Liberman, Tieder, and associates [176,180,183] have presented data that indicate the primary inborn error underlying this disorder is an expressed abnormality in the renal proximal tubule, which impairs phosphate reabsorption. They propose that this pivotal defect in turn stimulates renal 25(OH)D-1a-hydroxylase, thus

promoting the production of 1,25(OH)2D and increasing its serum and tissue levels. As a result intestinal calcium and phosphorus absorption is augmented and the renal filtered calcium load consequently increased. The enhanced intestinal calcium absorption also suppresses parathyroid function. In addition, prolonged hypophosphatemia diminishes osteoid mineralization, resulting in rickets and/or osteomalacia. The proposal that abnormal phosphate transport results in increased calcitriol production remains untested. Indeed, the elevation of 1,25(OH)2D in patients with HHRH is a unique phenotypic manifestation of the disease that distinguishes it from other disorders in which abnormal phosphate transport is likewise manifest. Such heterogeneity in the phenotype of these disorders suggests that disease at variable anatomical sites along the proximal convoluted tubule or involving compensatory enhancement of sodium-dependent phosphate cotransporters, such as that in the Npt2ae/e mouse [28,29], while uniformly impairing phosphate transport, may not necessarily inhibit 25(OH)D-1ahydroxylase activity. GENETIC DEFECT

Autosomal recessive transmission of HHRH seems consistent with the inheritance pattern in the described kindreds. However, if HHRH is an autosomal recessive disease and individuals with idiopathic hypercalciuria (IH) are heterozygous for the mutant allele, IH must be an incompletely penetrant trait because not all obligate heterozygotes manifest hypercalciuria. Alternatively, it has been suggested that HHRH and IH could be the result of mutations in two different genes [180]. The strongest support against this hypothesis is that when individuals with HHRH are treated with oral Pi, both the hypophosphatemia and the hypercalciuria are corrected. Moreover, recent studies have identified mutations in the SLC34A3 gene, coding the Npt2c protein, as causal of the HHRH, albeit the Npt2c protein was thought to have only a minor role in renal Pi transport. In fact, mice that are null for Npt2c (Npt2c/) reportedly show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. However, double knockout mice (Npt2a/ and Npt2c/) do exhibit a phenotype (hypophosphatemia, hypercalciuria, and rickets) substantially more severe than that of Npt2a and Npt2c knockout mice. Such observations lend credence to a substantial role for the Npt2c protein in the regulation of renal phosphate transport and maintenance of phosphate homeostasis [14]. TREATMENT

In accord with the hypothesis that a defect in renal phosphate transport alone underlies HHRH, patients

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have been treated successfully with high-dose phosphorus (1e2.5 g/d in five divided doses) alone. Within several weeks after initiation of therapy, bone pain disappears and muscular strength improves substantially. Moreover, the majority of treated subjects exhibit accelerated linear growth and radiological signs of rickets are completely absent within 4e9 months. Concordantly, serum phosphorus values increase towards normal, the 1,25(OH)2D concentration decreases, and alkaline phosphatase activity declines to normal. Despite this favorable response, limited investigation indicates that the osteomalacic component of the bone disease does not exhibit normalization. Further studies will be necessary, therefore, to determine if phosphorus alone will be sufficient treatment for this rare disorder. In any case, administration of phosphorus in patients with this disease does not result in the same spectrum of complications encountered upon its use in other disorders. Most notably, nephrocalcinosis, a common complication in treated patients with XLH, occurs infrequently in subjects with HHRH. In fact, the rare occurrence of this complication is associated with a history of vitamin D intoxication prior to initiation of treatment with phosphorus. Similarly, the development of secondary hyperparathyroidism in treated patients with HHRH has not been reported, although expectation of this complication is high since oral administration of phosphate does diminish the circulating calcium concentration and, in turn, stimulates parathyroid function. Fanconi Syndrome (FS) Rickets and osteomalacia are frequently associated with Fanconi syndrome, a disorder characterized by hyperphosphaturia and consequent hypophosphatemia, hyperaminoaciduria, renal glycosuria, albuminuria, and proximal renal tubular acidosis [184e187]. Damage to the renal proximal tubule, secondary to genetic disease or environmental toxins, represents the common underlying mechanism of this disease [188]. Resultant dysfunction causes renal wasting of those substances primarily reabsorbed at the proximal tubule. The inherited form may occur in isolation (in the absence of any other metabolic disease) or secondary to various primary Mendelian diseases. The associated bone disease in this disorder is likely secondary to hypophosphatemia and/or acidosis, abnormalities that occur in association with aberrantly (Fanconi syndrome, type I) or normally regulated (Fanconi syndrome, type II) vitamin D metabolism. FANCONI SYNDROME, TYPE I

Renal phosphate wasting and hypophosphatemia are the hallmark abnormalities of this disease, which

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resembles in many respects the more common genetic disease, XLH. In this regard, occurrence of abnormal bone mineralization appears dependent upon the prevailing renal phosphate wasting and resultant hypophosphatemia. Indeed, disease subtypes in which isolated wasting of amino acids, glucose, or potassium occur, are not associated with rickets and/or osteomalacia. Further, in the majority of patients studied, affected subjects exhibit abnormal vitamin D metabolism, characterized by serum 1,25(OH)2D levels that are overtly decreased or abnormally low relative to the prevailing serum phosphorus concentration [189e191]. Although the aberrantly regulated calcitriol biosynthesis may be due to the abnormal renal phosphate transport, as suspected in patients with XLH, proximal tubule damage and acidosis may play important roles. A notable difference between this syndrome and XLH is a prevailing acidosis, which may contribute to the bone disease. In this regard, several studies indicate that acidosis may exert multiple deleterious effects on bone. Such negative sequelae are related to the loss of bone calcium that occurs secondary to calcium release for use in buffering [184,192]. Alternatively, several investigators [193,194] have reported that acidosis may impair bone mineralization secondary to direct inhibition of renal 1a-hydroxylase activity. Others dispute these findings and claim that acidosis does not cause rickets or osteomalacia in the absence of hypophosphatemia. Indeed, Brenner et al. [195] reported that the rachitic/osteomalacic component of this disorder occurs only in patients with type 2 renal tubular acidosis and phosphate wasting. In contrast, those with types 1 and 4 renal tubular acidosis displayed no evidence of abnormal bone mineralization. Thus, the interplay of acidosis and phosphate depletion on bone mineralization in this disorder remains poorly understood. Most likely, however, hypophosphatemia and abnormally regulated vitamin D metabolism are the primary factors underlying rickets and osteomalacia in this form of Fanconi syndrome. Although the genetic defect resulting in this disease has been unknown, recent studies have identified a homozygous duplication in SLC34A1, encoding Npt2a, as the cause of autosomal recessive Fanconi syndrome associated with hypophosphatemic rickets and renal failure. This finding provides a human model for the loss of function of Npt2a, constitutes previously unavailable evidence for the inclusion of Npt2a in the list of proteins involved in human hypophosphatemic rickets and proximal tubulopathy, and validates the pivotal role of the human Npt2a cotransporter in renal phosphate handling and in the maintenance of wholebody phosphate homeostasis. The mechanism whereby intracellular accumulation of a membrane-targeted transporter such as Npt2a may induce a more

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generalized proximal-tubule dysfunction and renal failure is currently unknown but is likely to become the subject of further research [196]. FANCONI SYNDROME, TYPE II

Tieder et al. [197] have described two siblings (from a consanguineous mating) who presented with classic characteristics of Fanconi syndrome, including renal phosphate wasting, glycosuria, generalized aminoaciduria, and increased urinary uric acid excretion. However, these patients had appropriately elevated (relative to the decreased serum phosphorus concentration) serum 1,25(OH)2D levels and consequent hypercalciuria despite normal serum parathyroid hormone levels and cyclic AMP excretion. Moreover, treatment with phosphate reduced the serum calcitriol in these patients into the normal range and normalized the urinary calcium excretion. In many regards, this syndrome resembles HHRH and represents a variant of Fanconi syndrome, referred to as type II disease. The bone disease in affected subjects is likely due to the effects of hypophosphatemia. In any case, the existence of this variant form of disease is probably the result of renal damage to a unique segment of the proximal tubule or involvement of a different mechanism at the same site [197]. Further studies will be necessary to distinguish these possibilities. TREATMENT

Ideally, treatment of the bone disease in this disorder should offset the pathophysiological defect influencing proximal renal tubular function. In many cases, however, the primary abnormality remains unknown. Moreover, efforts to decrease tissue levels of causal toxic metabolites by dietary (such as in fructose intolerance) or pharmacological means (such as in cystinosis and Wilson syndrome) have met with variable success. Indeed, no evidence exists that indicates if the proximal tubule damage is reversible upon relief of an acute toxicity. Regardless, in instances when specific therapies are not available or do not lead to normalization of the primary defect, therapy must be directed at raising the serum phosphorus concentration, replacing calcitriol (in type I disease) and reversing the associated acidosis. However, use of phosphorus and calcitriol in this disease has been limited. In general, such replacement therapy leads to substantial improvement or resolution of the bone disease [198]. Unfortunately, growth and developmental abnormalities, more likely associated with the underlying genetic or acquired disease, remain substantially impaired [198]. More efficacious therapy, therefore, is dependent upon future research into the causes of the multiple disorders that underlie this syndrome.

X-linked Recessive Hypophosphatemia (XLRH) The initial description of X-linked recessive hypophosphatemic rickets involved a family in which males presented with rickets or osteomalacia, hypophosphatemia, and a reduced renal threshold for phosphate reabsorption. In contrast to patients with XLH, affected subjects exhibited hypercalciuria, elevated serum 1,25 (OH)2D levels (Table 63.1), and proteinuria of up to 3 g/day. Patients also developed nephrolithiasis and nephrocalcinosis with progressive renal failure in early adulthood. Female carriers in the family were not hypophosphatemic and lacked any biochemical abnormalities other than hypercalciuria. Three related syndromes have been reported independently: X-linked recessive nephrolithiasis with renal failure, Dent’s disease, and low-molecular-weight proteinuria with hypercalciuria and nephrocalcinosis. These syndromes differ in degree from each other, but common themes include proximal tubular reabsorptive failure, nephrolithiasis, nephrocalcinosis, progressive renal insufficiency, and, in some cases, rickets or osteomalacia. Identification of mutations in the voltage-gated chloride-channel gene CLCN5 in all four syndromes has established that they are phenotypic variants of a single disease and are not separate entities [199,200]. However, the varied manifestations that may be associated with mutations in this gene, particularly the presence of hypophosphatemia and rickets/osteomalacia, underscore that environmental differences, diet, and/or modifying genetic backgrounds may influence phenotypic expression of the disease.

Disorders of Renal Phosphate Transport: Hyperphosphatemic Diseases (Hyperphosphatemic) Tumoral Calcinosis (TC) Tumoral calcinosis is a rare genetic disease characterized by periarticular cystic and solid tumorous calcifications. Most patients in North America with this disorder are black and about one-third of the cases are familial. There is no gender preference. Biochemical markers of the disorder include hyperphosphatemia and an elevated serum 1,25(OH)2D concentration in many patients. Using these criteria, evidence has been presented for autosomal recessive inheritance of this syndrome. The hyperphosphatemia characteristic of the disease results from an increase in capacity of renal tubular phosphate reabsorption [201,202]. Hypocalcemia is not a consequence of this abnormality, however, and the serum parathyroid hormone concentration is normal. Moreover, the phosphaturic and urinary cAMP responses to parathyroid hormone are not disturbed.

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Interestingly, affected patients manifest increased circulating 1,25(OH)2D levels despite hyperphosphatemia, underlining the fact that it is TmP/GFR rather than the serum phosphorus concentration that controls 1a-hydroxylase activity (Fig. 63.1). Undoubtedly, the calcific tumors result from the elevated calciumephosphorus product. The observation that long-term phosphorus depletion alone [203] or in association with administration of aluminum hydroxide [204] or acetazolamide, a phosphaturic agent [205], leads to resolution of the tumor masses supports this possibility. Furthermore, reduction of phosphate levels in extracellular fluid helps prevent reformation of mineral deposits [203]. In addition, preliminary studies indicate that calcitonin therapy may also be efficacious by enhancing phosphaturia [206]. An acquired form of this disease is rarely seen in patients with end-stage renal failure. Affected patients manifest hyperphosphatemia in association with either: (1) an inappropriately elevated calcitriol level for the degree of renal failure, hyperparathyroidism or hyperphosphatemia; or (2) long-term treatment with calcium carbonate, calcitriol, or high-calcium-content dialysates. Again, calcific tumors likely result from an elevated calciumephosphorus product. Indeed, complete remission of the tumors occurs with treatment with vinpocetine, a mineral scavenger drug. A series of recent studies have documented that the autosomal recessive disorder is due to loss-of-function mutations in FGF23, as well as GALNT3 and Klotho, all causing familial tumoral calcinosis because of inadequate intact FGF23 concentrations or action. FGF23 MUTATIONS

It is not surprising that a genetic defect underlying familial tumoral calcinosis is an FGF23 loss-of-function mutation, since the phenotype of the disorder is the metabolic mirror image of that in patients with ADHR, which results from a gain-of-function mutation. Indeed, several reports have documented that affected individuals with familial tumoral calcinosis had biallelic mutations in the FGF23 gene [207e210]. The mutations resulted in various abnormalities including: (1) destabilization of full-length FGF23; and (2) retention of the fulllength FGF23 in the Golgi complex and secretion of only the biologically inactive C-terminal fragment. Hence, the syndrome resembles that evident in FGF23 knockout mice. GALNT3 MUTATIONS

Novel biallelic mutations affecting UDP-N-acetyl-aD-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc transferase 3) have been found in patients of Middle Eastern and European descent [211e213]. This protein is a Golgi-associated biosynthetic

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enzyme, which initiates mucin-type O-glycosylation of proteins. O-glycosylation of FGF23 by the GalNAc transferase 3 is essential for the secretion of intact FGF23 because O-glycosylation at a subtilisin-like proprotein convertase recognition sequence motif prevents cleavage of FGF23. The absence of secretion underlies the loss-offunction nature of the mutation. In these studies of patients with GALNT3 mutations have indicated phenotypic variability in affected individuals. While the mutations invariably result in hyperphosphatemia as a result of low circulating FGF23 concentrations, other clinical manifestations of the disorder were variable. Indeed, the spectrum of phenotypic abnormalities ranged from those typical of familial tumoral calcinosis to those associated with hyperostosisehyperphosphatemia (recurrent long bone lesions with hyperostosis). Therefore, tumoral calcinosis and hyperostosisehyperphosphatemia syndrome likely represent a continuous spectrum of the same disease caused by increased phosphate levels, rather than two distinct disorders. KLOTHO MUTATIONS

Recently Ichikawa et al. [214] reported a homozygous missense mutation in the KLOTHO gene in a patient who presented with severe tumoral calcinosis. The mutation attenuated production of the membrane bound Klotho, resulting in diminished ability of FGF23 to signal via its cognate FGFR1 receptors. As a consequence, FGF23 resistance resulted in a syndrome reflecting absent FGF23 action. See Chapter 42 for further discussion of the role of Klotho in FGF23 action. The genetic abnormalities underlying familial tumoral calcinosis add to the evolving story of the role that FGF23 plays in the evolution of genetic disorders of hypophosphatemic and hyperphosphatemic diseases. Indeed, the data available argue forcefully for the central role of this phosphatonin in the pathophysiology of these diseases.

DISORDERS RELATED TO AN ALTERED PHOSPHATE LOAD Decreased Phosphate Load Phosphate Deprivation Hypophosphatemia and phosphate depletion due to inadequate dietary intake are rare. However, studies indicate that hypophosphatemia is a relatively frequent occurrence in children under 5 years of age presenting with kwashiorkor or marasmic kwashiorkor [215]. With a decline in ingested phosphate, the renal TmP increases and urinary phosphate excretion decreases [216]. In addition, gastrointestinal phosphate secretion gradually lessens. However, severe dietary deprivation

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(less than 100 mg/d) leads to a prolonged period of negative phosphate balance and total body depletion. Affected females may display hypophosphatemia (1.4 to 2.5 mg/dl). Interestingly, in contrast, males generally do not manifest a decreased serum phosphate concentration in response to dietary deprivation. Nevertheless, attempts to maintain phosphate homeostasis in both sexes include suppression of the serum PTH concentration and increased 1,25(OH)2D production by an unknown mechanism. Thus, hypercalciuria may be associated with the syndrome. Whether the efficiency of calcitriol responsiveness or differential effects on end organs, such as the gastrointestinal tract or bone, underlies the noted gender difference remains unclear. Total starvation does not cause profound hypophosphatemia. The catabolic effects of total food deprivation result in the release of phosphate from intracellular stores, which compensates for the negative phosphorus balance. However, refeeding of the starved person can precipitate hypophosphatemia if phosphate deprivation is maintained. Indeed, this “refeeding syndrome” was observed by Kimutai et al. [215] when patients with phosphate depletion due to kwashiorkor and marasmic kwashiorkor received nutritional intervention. A decrease in serum phosphorus levels to levels as low as 0.63 mmol/liter (~2.0 mg/dl) occurred, resulting in deaths amongst 15% of the population. Gastrointestinal Malabsorption Gastrointestinal absorption of phosphorus may be decreased with the use of aluminum- or magnesiumcontaining antacids, as well as other drugs, including calcium acetate/magnesium carbonate and sevelamer hydrochloride; prolonged use of these drugs in large amounts has been associated with hypophosphatemia and a negative phosphorus balance [217]. Long-term reduction of the serum phosphorus concentration owing to chronic, excessive use of antacids leads to frank osteomalacia and myopathy. The osteomalacia results as a direct consequence of the phosphate depletion and in spite of normal vitamin D stores and an increased serum 1,25(OH)2D concentration, which occurs in response to the hypophosphatemia. In contrast, the pathophysiology of variably occurring osteomalacia secondary to gastrointestinal diseases that cause steatorrhea or rapid transit time (e.g., Crohn’s disease, postgastrectomy states, and intestinal fistulas) is significantly different [218]. In this case, mild to moderate hypophosphatemia occurs due to vitamin D malabsorption/deficiency and resultant secondary hyperparathyroidism and renal phosphate wasting. Further, the relation between the metabolic bone disease, vitamin D deficiency, and hypophosphatemia is

complex and likely involves the influence of phosphopenia and vitamin-D-dependent calciopenia on bone mineralization. However, the impact of vitamin D deficiency may be overriding since osteomalacia may occur in the absence of hypophosphatemia. Regardless, in the presence of hypophosphatemia (and/or phosphate depletion), an elevated serum calcitriol level is part of this syndrome, likely due to elevated serum parathyroid hormone and has no apparent effect on the evolution of the bone disease. Many other causes of malabsorption may likewise influence phosphate and vitamin D homeostasis. See Chapter 69 for a review of these GI diseases.

Transcellular Shift In a large proportion of clinically important cases of hypophosphatemia, a sudden shift of phosphorus from the extracellular to the intracellular compartment is responsible for the decline of the serum phosphate concentration. This ion movement occurs in response to naturally occurring disturbances and after the administration of certain compounds. Alkalosis Alkalosis secondary to intense hyperventilation may depress serum phosphate levels to less than 1 mg/dl [219]. A similar degree of alkalemia from excess bicarbonate also causes hypophosphatemia, but of a much lesser magnitude (2.5 to 3.5 mg/dl). The disparity between the effects of a respiratory and metabolic alkalosis is related to the more pronounced intracellular alkalosis that occurs during hyperventilation. The phosphate shift to the intracellular compartment results from the utilization attendant on glucose phosphorylation, a process stimulated by a pH-dependent activation of phosphofructokinase. Glucose Administration The administration of glucose and insulin often results in moderate hypophosphatemia [220]. Endogenous or exogenous insulin increases the cellular uptake not only of glucose, but also of phosphorus. The most responsive cells are those of the liver and skeletal muscle. The decline of the serum phosphate concentration generally does not exceed 0.5 mg/dl. A lesser decrease is manifest in patients with type 2 diabetes mellitus and insulin resistance or those with a disease causing a diminished skeletal mass. The administration of fructose and glycerol similarly reduces the serum phosphorus concentration. In contrast to glucose, fructose administration may be associated with more pronounced hypophosphatemia; the more striking effect is due to unregulated phosphorus uptake by the liver.

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Combined Mechanisms There are special clinical situations in which an altered phosphate and consequent hypophosphatemia result from both a transcellular shift of phosphorus and phosphate deprivation or renal phosphate wasting. These disorders represent some of the more common and profound causes of a decreased serum phosphorus concentration. Alcoholism Alcoholic patients frequently enter the hospital with hypophosphatemia. However, many do not exhibit a decreased serum phosphate concentration until several days have elapsed and refeeding has begun. The multiple factors that underlie the hypophosphatemia include poor dietary intake, use of phosphate binders to treat gastritis, excessive urinary losses of phosphorus, and shift of phosphorus from the extracellular to the intracellular compartment, owing to glucose administration and/or hyperventilation that occurs in patients with cirrhosis or during alcohol withdrawal [220]. Moreover, many alcoholic patients are hypomagnesemic, which potentiates renal phosphate wasting by an unclear mechanism. Burns Within several days after sustaining an extensive burn, patients often manifest severe hypophosphatemia. The initial insult induces a transient retention of salt and water. When the fluid is mobilized, significant urinary phosphorus loss ensues. Coupled with the shift of phosphorus to the intracellular compartment, which occurs secondary to hyperventilation, and the anabolic state, profound hypophosphatemia may result. Nutritional Recovery Syndrome Refeeding of starved individuals or maintaining nutritional support by parenteral nutrition or by tube feeding, without adequate phosphorus supplementation, may also cause hypophosphatemia [221]. A prerequisite for the decreased serum phosphate concentration in affected patients is that their cells must be capable of an anabolic response. As new proteins are synthesized and glucose is transported intracellularly, phosphate demand depletes reserves. Several days are generally required after the initiation of refeeding in order to establish an anabolic condition. In patients receiving total parenteral nutrition, serum phosphate levels may be further depressed if sepsis supervenes and a respiratory alkalosis develops. Diabetic Ketoacidosis Poor control of blood glucose and consequent glycosuria, polyuria, and ketoacidosis invariably cause

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renal phosphate wasting [220]. The concomitant volume contraction may yield a normal serum phosphate concentration. However, with insulin therapy, the administration of fluids, and correction of the acidosis, serum and urine phosphate fall precipitously. The resultant hypophosphatemia may contribute to insulin resistance and slow the resolution of the ketoacidosis. Hence, the administration of phosphate may improve the capacity to metabolize glucose and facilitate recovery.

Increased Phosphate Load Vitamin D Intoxication An increase of the phosphate load from exogenous sources generally does not cause hyperphosphatemia because the excessive phosphorus is excreted by the kidney. However, an increased serum phosphate concentration may occur in vitamin D intoxication when the gastrointestinal absorption of phosphate is markedly enhanced. Increased phosphate mobilization from bone and a reduction of GFR, secondary to hypercalcemia and/or nephrocalcinosis, may also contribute to the evolution of the hyperphosphatemia. The chronic ingestion of large doses of vitamin D, in excess of 100 000 IU/day, is generally required to cause intoxication. Suspected hypervitaminosis D may be investigated using specific assays, which can document excessive amounts of vitamin D and its metabolites in the circulation (see Chapters 45 and 72 for a discussion of vitamin D intoxication). Rhabdomyolysis Because muscle contains a large amount of phosphate, necrosis of muscle tissue may acutely increase the endogenous phosphate load and result in hyperphosphatemia. Such muscle necrosis (rhabdomyolysis) may complicate heat stroke, acute arterial occlusion, hyperosmolar non-ketotic coma, trauma, toxic agents such as ethanol and heroin, and idiopathic paroxysmal myoglobinuria [222,223]. Muscle biopsy often reveals myolytic denervation. As a consequence of muscle destruction, acute renal failure caused by myoglobin excretion frequently complicates the clinical presentation and contributes to the hyperphosphatemia. However, an elevated serum phosphate concentration may precede evidence of renal failure, or occur in its complete absence when rhabdomyolysis is present. The diagnosis is confirmed by elevated serum creatine phosphokinase, uric acid, and lactate dehydrogenase concentrations, and the demonstration of heme-positive urine in the absence of red blood cells. Therapy is directed at the underlying disorder with maintenance of the extracellular volume to avoid volume depletion

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and alkalinization of the urine to prevent uric acid accumulation and consequent acute tubular necrosis. Cytotoxic Therapy Cytotoxic therapy often causes cell destruction and liberation of phosphorus into the circulation [224]. The lysis of tumor cells begins within 1 to 2 days after initiating treatment, and is followed quickly by an elevation of the serum phosphate concentration. Hyperphosphatemia supervenes, however, only when the treated malignancies have a large tumor burden, rapid cell turnover, and substantial intracellular phosphorus content. Such malignancies include lymphoblastic leukemia, various types of lymphoma, and acute myeloproliferative syndromes. Hyperkalemia and hyperuricemia also occur. Indeed, uric acid nephropathy may cause renal insufficiency that predisposes to further phosphate retention and worsening of the hyperphosphatemia. Malignant Hyperthermia Malignant hyperthermia is a rare familial syndrome characterized by an abrupt rise in body temperature during the course of anesthesia [225]. The disease appears to be autosomal dominant in transmission, and an elevated serum creatine phosphokinase concentration is found in otherwise normal family members. Hyperphosphatemia results from shifts of phosphate from muscle cells to the extracellular pool. A high mortality rate accompanies the syndrome.

Clinical Signs and Symptoms of Abnormal Serum Phosphorus in Diseases Caused by an Altered Phosphate Load As related above, a wide variety of diseases and syndromes with varying clinical manifestations have the characteristic biochemical abnormalities of hyperphosphatemia or hypophosphatemia. A unique complex of disturbances often is directly related to the abnormal phosphate homeostasis. The recognition of these signs and symptoms may lead to appropriate biochemical testing, the diagnosis of an unsuspected disease, and initiation of lifesaving or curative treatment. Hypophosphatemia Hypophosphatemia secondary to impaired expression or function of phosphate transporters frequently causes renal stones in affected mice and humans [5]. Indeed, a decreased TmP/GFR (<0.70 mmol/liter), indicative of impaired renal phosphate transport, is present in 20% of subjects in whom renal stones form but in only 5% of control subjects [226]. Renal phosphate

wasting is also associated with bone demineralization as evident in innumerable diseases discussed above. More profound consequences of a low serum phosphorus level occur if the hypophosphatemia is associated with concomitant phosphate depletion. Such phosphate deficiency may cause widespread disturbances. This is not surprising, since severe hypophosphatemia/phosphate deficiency causes two critical abnormalities that impact on virtually all organ systems. First, a deficiency of 2,3-diphosphoglycerate (2,3-DPG) occurs in red cells, which is associated with an increased affinity of hemoglobin for oxygen and, therefore, tissue hypoxia. Second, there is a decline of tissue ATP content and a concomitant decrease in the availability of energyrich phosphate compounds that are essential for cell function [227,228]. The major clinical syndromes resulting from these abnormalities include nervous system dysfunction, anorexia, nausea, vomiting, ileus, muscle weakness, cardiomyopathy, respiratory insufficiency, hemolytic anemia, and impaired leukocyte and platelet function. Additionally, phosphate deficiency causes osteomalacia and bone pain, clinical sequelae that are probably independent of the aforementioned abnormalities. Central nervous system dysfunction has been well characterized in severe hypophosphatemia/phosphate depletion, especially in patients receiving total parenteral nutrition for diseases causing severe weight loss. A sequence of symptoms compatible with a metabolic encephalopathy usually begins one or more weeks after the initiation of therapy with solutions that contain glucose and amino acids, but lack adequate phosphorus supplementation to prevent hypophosphatemia. The onset of dysfunction is marked by irritability, muscle weakness, numbness, and paresthesias, with progression to dysarthria, confusion, obtundation, coma, and death [229]. These patients have a profoundly diminished red cell 2,3-DPG content. Both biochemical abnormalities and clinical symptoms improve as patients receive phosphorus supplementation. Peripheral neuropathies, Guillain-Barre-like paralysis, hyporeflexia, intention tremor, and ballismus have also been described with hypophosphatemia and phosphate depletion. The effects of hypophosphatemia on muscle depend on the severity and chronicity of the deficiency. Chronic phosphorus deficiency results in a proximal myopathy with striking atrophy and weakness. Osteomalacia frequently accompanies the myopathy, so patients complain of pain in weight-bearing bones. Normal values for serum creatine phosphokinase and aldolase activities are characteristically present. Rhabdomyolysis does not occur with chronic phosphate depletion. In contrast, acute hypophosphatemia can lead to rhabdomyolysis with muscle weakness and pain. Most

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DISORDERS RELATED TO AN ALTERED PHOSPHATE LOAD

cases occur in chronic alcoholics or patients receiving total parenteral nutrition. In both groups of patients, muscle pain, swelling, and stiffness occur 3 to 8 days after the initiation of therapies that do not contain adequate amounts of phosphorus. Muscle paralysis and diaphragmatic failure may occur. Studies of muscle tissue from chronically phosphate-depleted dogs made acutely hypophosphatemic show a decrement in cellular content of phosphorus, ATP, and adenosine diphosphate (ADP). Rhabdomyolysis occurred in these muscle fibers. The laboratory findings in patients with hypophosphatemic myopathy and rhabdomyolysis include elevated serum creatine phosphokinase levels; however, serum phosphate levels may become normal if enough necrosis has occurred with subsequent phosphorus release. Also, renal failure and hypocalcemia can be associated with the syndrome. Myocardial performance can be abnormal at serum phosphate levels of 0.7 to 1.4 mg/dL, which reflect profound phosphate depletion. This occurs when ATP depletion causes impairment of the actinemyosin interaction, the calcium pump of the sarcoplasma, and the sodiumepotassium pump of the cell membrane [230]. The net result is reduced stroke work and cardiac output, which may progress to congestive heart failure. These problems are reversible with phosphate replacement. Respiratory failure can occur owing to failure of diaphragmatic contraction in hypophosphatemic patients. When serum phosphate levels are raised, diaphragmatic contractility improves. The postulated mechanism for the respiratory failure is muscle weakness secondary to inadequate levels of ATP and decreased glycolysis as the result of phosphate depletion [231]. Two disturbances of red cell function may occur secondary to phosphorus deficiency. First, as intraerythrocyte ATP production is decreased, the erythrocyte cell membrane becomes rigid, which can cause hemolysis [232]. This is rare and is usually seen in septic, uremic, acidotic, or alcoholic patients, when serum phosphate levels are less than 0.5 mg/dl. Second, the limited production of 2,3-DPG causes a leftward shift of the oxyhemoglobin curve and impairs the release of oxygen to peripheral tissues. Such a consequence of chronic hypophosphatemia has been documented in children with XLH and proposed as one factor underlying retarded growth stature [233]. Leukocyte dysfunction, which complicates phosphate deficiency, includes decreased chemotaxis, phagocytosis, and bactericidal activity [234]. These abnormalities increase the host susceptibility to infection. The mechanism by which hypophosphatemia impairs the various activities of the leukocyte probably is related to impairment of ATP synthesis. Decreased availability of energy impairs microtubules that regulate

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the mechanical properties of leukocytes and limits the rate of synthesis of organic phosphate compounds that are necessary for endocytosis. Abnormal platelet survival, causing thrombocytopenia, profuse gastrointestinal bleeding, and cutaneous bleeding, has also been described in association with phosphate depletion in animal studies. Despite these abnormalities, there is little evidence that hypophosphatemia is a primary cause of hemorrhage in humans. Perhaps the most consistent abnormalities associated with phosphate depletion are those on bone. Acute phosphate depletion induces dissolution of apatite crystal from the osseous matrix. This effect may be due to 1,25(OH)2D3, which is increased in response to phosphate depletion in both animals and humans. More prolonged hypophosphatemia leads to rickets and osteomalacia. This complication is a common feature of phosphate depletion. However, the ultimate cause is variable. While simple phosphate depletion alone may underlie the genesis of the abnormal mineralization, in many disorders the defect is secondary to phosphate depletion and commensurate 1,25(OH)2D3 deficiency. Thus, treatment of this complication may often require combination therapy, phosphate supplements, and calcitriol. Hyperphosphatemia Hypocalcemia and consequent tetany are the most serious clinical sequelae of hyperphosphatemia [235]. The decreased serum calcium concentration results from the deposition of calcium phosphate salts in soft tissue, a process that may lead to symptomatic ectopic calcification. The dystrophic calcification is frequently seen in acute and chronic renal failure, hypoparathyroidism, pseudohypoparathyroidism, and tumoral calcinosis. Indeed, deposition of calciumephosphate complexes in the kidney may predispose to acute renal failure. When the calciumephosphate product exceeds 70, the probability that soft tissue calcification will occur increases sharply. In addition, local factors, such as tissue pH and injury (e.g., necrotic or hypoxic tissue), may predispose to precipitation of the calciumephosphate salts. In chronic renal failure, calcification occurs in arteries, muscle tissue, periarticular spaces, the myocardial conduction system, lungs, and the kidney. Affected patients may also have ocular calcification, causing the “red eye” syndrome of uremia and subcutaneous calcification, which also plays a role in uremic pruritus. On the other hand, a predisposition to calcification of periarticular surfaces of the hips, elbows, shoulders, and other large joints occurs in tumoral calcinosis. In some disease states, hyperphosphatemia may also play an important role in the development of secondary hyperparathyroidism [236]. A decrement in the serum calcium concentration secondary to hyperphosphatemia

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stimulates the release of PTH. Furthermore, hyperphosphatemia decreases the activity of 1a-hydroxylase. The consequent diminished production of 1,25(OH)2D3 impairs the gastrointestinal absorption of calcium and induces skeletal resistance to PTH, influences that augment the development of hyperparathyroidism. Thus, hyperphosphatemia triggers a cascade of events that impact on calcium homeostasis at multiple sites. Therefore, the prevention of secondary hyperparathyroidism, metabolic bone disease, and soft tissue and vascular calcification in affected patients depends on ultimately controlling the serum phosphate concentration.

Treatment of Abnormal Serum Phosphorus in Diseases Caused by an Abnormal Phosphate Load Treatment of the myriad of diseases that characteristically display hyperphosphatemia or hypophosphatemia depends on determining the mechanism underlying their pathogenesis. The cause can almost always be ascertained by assessment of the clinical setting, determination of renal function, measurement of urinary phosphate excretion, and analysis of arterial carbon dioxide tension and pH. Therapy is aimed at correcting both the serum phosphate concentration and associated complications. The treatment of phosphate depletion depends on replacing body phosphorus stores. Preventive measures, however, will preclude the onset of phosphate depletion. Thus, appropriate monitoring of patients taking large doses of aluminum-containing antacids and provision of phosphorus intravenously to patients with diabetic ketoacidosis will preserve phosphate stores. Alternatively, the treatment of established phosphate depletion may require 2.5 to 3.7 g of phosphate daily, preferably administered orally in at least four equally divided doses. Providing K-Phos Neutral tablets, which contain 250 mg of elemental phosphorus per tablet, will fulfill this goal. If oral therapy is not tolerated and the serum phosphate shows a downward trend approaching dangerous levels (<1.2 mg/dl), intravenous phosphate supplementation at a dose of 10 mg/kg body weight/day may be administered. Such therapy should be discontinued when the serum phosphate reaches values >2.0 mg/dl. However, therapy is not required for many of the conditions resulting in phosphate depletion. Only when the consequences of severe depletion are manifest should treatment be initiated. Theoretically, treatment of an elevated phosphate load and lowering the attendant serum phosphorus concentration depend upon decreasing the TmP, increasing the GFR, or diminishing the phosphate load

(exogenous or endogenous). There are no generally available pharmacologic means of acutely altering the GFR or reducing the TmP. However, chronic use of drugs, such as acetazolamide, which decreases TmP and induces phosphaturia, is effective as ancillary treatment of disorders such as tumoral calcinosis. More recently, attention has turned to increasing endogenous FGF23 or providing pharmacological preparations to lower the serum phosphorus over prolonged periods of time. Nevertheless, regulation of hyperphosphatemia is most often achieved by reducing the renal phosphate load. In tumoral calcinosis and chronic renal failure, such an effect is obtained by restricting the dietary phosphate intake or by administering phosphate binders. Alternative strategies for management of load-dependent hyperphosphatemia include the administration of intravenous calcium or intravenous glucose and insulin. The consequence of such intervention is sequestration of phosphate in bone or soft tissues. Dialysis can also be used for the acute management of load-dependent disorders, or the chronic maintenance of phosphate overload such as those that complicate chronic renal failure. The problem of phosphate overload in renal disease is discussed in Chapter 70.

CONCLUDING REMARKS Over the past several decades it has become abundantly clear that an alteration of phosphate homeostasis can impact the function of a wide variety of tissues. As the control mechanisms for phosphate homeostasis have been discovered, specific abnormalities in phosphate transport have been identified and linked to a variety of diseases. Particular evolution of concepts regarding the role of phosphate in human bone diseases has been quite remarkable. Indeed, the similarities between various bone diseases can now be traced to variable mechanisms that alter the regulatory factors governing renal phosphate reabsorption. Perhaps most importantly, the advances recorded in understanding this regulatory process have led to emerging concepts for potentially effective treatment strategies. The enhanced information regarding diseases of phosphate homeostasis has also charted a better understanding of disorders related to an altered phosphate load and the potential consequences of such disorders. As a result appreciation of the available treatment strategies has improved and the therapy offered for these disorders is currently more effective. The material offered in this chapter is intended not only to provide up-to-date information but to begin formulating central theses and hypotheses into which forthcoming information is integrated.

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REFERENCES

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