p hosphorus homeostasis and related disorders · the average dietary phosphate intake in humans,...

Principles of Bone Biology, 3rd Edition

Copyright © 2008 by Academic Press. Inc. All rights of reproduction in any form reserved. 465

Chapter 1

Phosphorus plays an important role in cellular physiol-

ogy and skeletal mineralization, serving as a constituent

of nucleic acids and hydroxyapatite, a source of the high-

energy phosphate in adenosine triphosphate, an essential

element of the phospholipids in cell membranes, and a fac-

tor influencing a variety of enzymatic reactions (e.g., gly-

colysis) and protein functions (e.g., the oxygen-carrying

capacity of hemoglobin by regulation of 2,3-diphospho-

glycerate synthesis). Indeed, phosphorus is one of the most

abundant components of all tissues, and disturbances in

phosphate homeostasis can affect almost any organ sys-

tem. Most phosphorus within the body is in bone (600–

700 g); the remainder is largely distributed in soft tissue

(100–200 g). As a consequence, less than 1% of the total

is in extracellular fluids. The plasma contains about 12 mg/

dL of phosphorus, of which approximately 8 mg is organic

and contained in phospholipids, a trace is an anion of pyro-

phosphoric acid, and the remainder is inorganic phosphate

(P i ) ( Yanagawa et al. , 1994 ). Inorganic phosphate is pres-

ent in the circulation as divalent monohydrogen phosphate

and monovalent dihydrogen phosphate. At normal pH, the

relative concentrations of monohydrogen and dihydrogen

phosphate are 4:1.

The critical role that phosphorus plays in cell physiol-

ogy has resulted in development of elaborate mechanisms

designed to maintain phosphate balance. These adaptive

changes are manifest by a constellation of measurable

responses, the magnitude of which is modified by the differ-

ence between metabolic P i need and exogenous P i supply.

Such regulation maintains plasma and extracellular fluid

phosphorus within a relatively narrow range and depends

primarily on gastrointestinal absorption and renal excretion

as mechanisms to affect homeostasis. Although investiga-

tors have recognized a variety of hormones that influence

Chapter 23

these various processes, in concert with associated changes

in other metabolic pathways, the sensory system, the mes-

senger, and the mechanisms underlying discriminant regu-

lation of P i balance remain incompletely understood.

Whereas long-term changes in P i balance depend on

these variables, short-term changes in phosphate concen-

trations can occur as a result of redistribution of phosphate

between the extracellular fluid and either bone or cell con-

stituents. Such redistribution results secondary to various

mechanisms, including elevated levels of insulin and/or

glucose; increased concentrations of circulating catechol-

amines; respiratory alkalosis; enhanced cell production or

anabolism; and rapid bone remineralization. In many of

these circumstances, hypophosphatemia manifests in the

absence of phosphorus depletion or deprivation.

REGULATION OF PHOSPHATE

HOMEOSTASIS

Phosphate is sufficiently abundant in natural foods that

phosphate deficiency is unlikely to develop except under

conditions of extreme starvation, as a consequence of

administration of phosphate binders, or secondary to

renal phosphate wasting. Indeed, the major proportion of

ingested phosphate is absorbed in the small intestine, and

hormonal regulation of this process plays only a minor role

in normal phosphate homeostasis. In contrast, absorbed

phosphate, in response to complex regulatory mechanisms,

is eliminated by the kidney, incorporated into organic

forms in proliferating cells, or deposited as calcium-phos-

phate complexes in soft tissue and/or as a component of

bone mineral (hydroxyapatite). The vast majority of the

absorbed phosphate, however, is excreted in the urine.

Thus, under usual conditions, phosphate homeostasis

depends for the most part on the renal mechanisms that

regulate tubular phosphate transport. Alternatively, dur-

ing times of severe phosphate deprivation, the phosphate

contained in bone mineral provides a source of phosphate

Phosphorus Homeostasis and Related Disorders

Marc K. Drezner*

Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism, University of Wisconsin, Madison, Wisconsin 53792

*Corresponding author: Marc K. Drezner, M.D. 4246 Health Sciences

Learning Center, University of Wisconsin, School of Medicine and Public

Health, 750 Highland Avenue, Madison, Wisconsin 53705. E-mail: mkd@

medicine.wise.edu

Part | I Basic Principles466

for the metabolic needs of the organism. The specific role

that the intestine and kidney play in this complex process

is discussed later.

Mechanism of Phosphate Transport

Cells obtain phosphorus in the form of negatively charged

P i from the extracellular environment by means of second-

ary-active transport. In vertebrates, P i transporters use the

inwardly directed electrochemical gradient of Na 1 ions,

established by the Na 1 ,K 1 -ATPase, to drive P i influx. Two

unrelated families of Na 1 -dependent P i transporters man-

age the active transport ( Virkki et al., 2007 ). The SCL34

or type II Na 1 /P i family prefer divalent HPO 4 2 2 and com-

prises both electrogenic and electroneutral members that

are expressed in various epithelia and other polarized cells.

Through regulated activity in apical membranes of the gut

and kidney, they maintain body P i homeostasis, and in sali-

vary and mammary glands, liver, and testes they play a role

in modulating the P i content of luminal fluids. The SLC20

or type III Na 1 /P i family transport monovalent H 2 PO 4 2

and consist of P i T-1 and P i T-2 receptors, which are elec-

trogenic and ubiquitously expressed, likely serving a

housekeeping role for cell P i homeostasis. However, more

specific roles are emerging for these transporters in bone P i

metabolism and vascular calcification.

The two families of Na 1 –P i cotransporters share no

significant homology in their primary amino acid sequence

and exhibit substantial variability in substrate affinity, pH

dependence, and tissue expression ( Table I ). The tissue

expression, relative renal abundance, and overall transport

characteristics of type II and III Na–P i cotransporters sug-

gest that the type II transporters play a key role in brush

border membrane P i flux ( Miyamoto et al., 2007 ).

The most detailed studies of the cellular events involved

in P movement from the luminal fluid to the peritubular

capillary blood have been performed in proximal tubules

and cultured cells derived from them. These investigations

indicate that P reabsorption occurs principally by a unidi-

rectional process that proceeds transcellularly with mini-

mal intercellular backflux from the plasma to the lumen.

Across the luminal membrane, P i entry into the tubular

cell proceeds by a saturable active transport system that is

sodium dependent ( Fig. 1 ).

The active transport of phosphate across the apical

membrane in renal proximal tubules is mediated by two

members of the SLC34 family of solute carriers, NaP i -IIa

( SLC34A1 ) and NaP i -IIc ( SLC34A3 ; see Table I ). These

receptors mediate the reabsorption of phosphate from

the urine by using the free energy provided by the elec-

trochemical gradient for Na 1 . NaP i -IIa is electrogenic

and transports divalent P i preferentially. It functions with

a Na 1 : P i stoichiometry of 3:1, which results in the net

movement inward of one positive charge per cotransport

cycle. In contrast, NaP i -IIc is electroneutral and exhibits a

2:1 stoichiometry. In mice, NaP i -IIa is the protein primarily

responsible for P i reabsorption in the adult kidney, whereas

TABLE I Summary of Known Parameters of the Type II and Type III Na/P i Transporters

Type II

Type IIa Type IIc Type IIb Type III

Chromosomal

location (human)

5 9 4 2 (P i T-1)

8 (P i T-2)

Amino acids , 640 601 , 690 679

656

Function Na–P i cotransport;

electrogenic, pH dependent

Na–P i cotransport;

neutral


electrogenic


electrogenic

Substrate P i P i P i P i

Affi nity for P i 0.1–0.2 m M 0.1–0.2 m M 0.05 m M 0.025 m M

Affi nity for Na 50–70 m M 50 m M 33 m M 40–50 m M

Na 1 –P i coupling 3 2 3 3

Tissue expression

(mRNA, protein)

Kidney, parathyroid Kidney Small intestine, lung,

other tissues

Ubiquitous

Major regulator(s) PTH, dietary P i , calcitriol,

FGF-23

PTH, dietary P i ,

FGF-23

Dietary P i , calcitriol, estrogen,

epidermal growth factor,

glucocorticoids

Dietary P i

FGF, fi broblast growth factor; PTH, parathyroid hormone.

467Chapter | 23 Phosphorus Homeostasis and Related Disorders

NaP i -IIc is seemingly more important in weanling animals.

The phenotype of NaP i -IIa knockout mice suggests that

that this cotransporter is responsible for the bulk of renal P i

reabsorption with a very small percentage potentially due

to the NaP i -IIc transporter. However, recent data indicate

that in humans, NaP i -IIc may have a previously unpre-

dicted importance (see the later subsection on Hereditary

Hypophosphatemic Rickets with Hypercalciuria:

Pathophysiology and Genetics). The expression of the

sodium-dependent phosphate cotransporters is regulated to

adapt the renal reabsorption of P i to the needs of the organ-

ism. Thus, the phosphaturic effects associated with para-

thyroid hormone or the phosphatonins, such as fibroblast

growth factor (FGF)-23, are due to the membrane retrieval

of both cotransporters, whereas in conditions of P i depriva-

tion their expression is increased.

The phosphate that enters the tubule cell plays a major

role in governing various aspects of cell metabolism and

function and is in rapid exchange with intracellular phos-

phate. Under these conditions, the relatively stable free P i

concentration in the cytosol implies that P i entry into the

cell across the brush border membrane must be tightly

coupled with its exit across the basolateral membrane

(see Fig. 1 ). The transport of phosphate across the baso-

lateral membrane is mediated by a transporter that remains

unidentified. Regardless, basolateral P i transport serves at

least two functions: (1) complete transcellular P i reabsorp-

tion when luminal P i entry exceeds the cellular P i require-

ments and (2) guaranteed basolateral P i influx if apical

P i entry is insufficient to satisfy cellular requirements

( Schwab et al. , 1984 ).

Gastrointestinal Absorption of Phosphorus

The average dietary phosphate intake in humans, derived

largely from dairy products, meat, and cereals, is 800 to

1600 mg/day, one and one-half to threefold greater than the

estimated minimum requirement. This phosphate is in both

organic and inorganic forms, but the organic forms, except

for phytates, are degraded in the intestinal lumen to inor-

ganic phosphate, which is the form absorbed. Absorption

occurs throughout the small intestine with transport great-

est in the jejunum and ileum and less in the duodenum.

Essentially no absorption occurs in the colon ( Walling,

1977 ).

The absorption of phosphorus in the intestine is depen-

dent on the amount and availability of phosphorus present

in the diet. In normal subjects, net P absorption is a linear

function of dietary P intake. Indeed, for a dietary P range

of 4 to 30 mg/kg/day, the net P absorption averages 60 to

65% of the intake ( Lee et al. , 1986 ). Intestinal P absorp-

tion occurs via two routes: transcellular and paracellular

pathways. Transcellular phosphorus flux is regulated by

a variety of hormones and metabolic factors, whereas the

paracellular pathway appears dependent on the magnitude

of the electrochemical gradients across the intestinal epi-

thelium. The latter transport pathway is still controversial

and may predominate at high intraluminal concentrations

of phosphorus, such as during the intake of a meal.

Transcellular intestinal absorption of phosphorus is ini-

tiated by the sodium-dependent transport of P i across the

apical (brush border) membrane. Phosphate incorporated

into intestinal cells by this mechanism is ferried from the

apical pole to the basolateral pole likely through restricted

channels such as microtubules. At the basolateral mem-

brane, phosphate is released from intestinal cells by a pas-

sive mechanism, which is carrier mediated and occurs in

accord with the electrochemical gradient. The apparent K m

for P i is between 0.1 and 0.2 m M , and lowering the external

pH value results in a modest increase of the transport rate.

The transepithelial transport of P i occurs predomi-

nantly in the small intestine and is determined largely by

the abundance of the Na/P i -IIb transporter in the brush

border membrane vesicles (see Table I ). Thus, regula-

tion of transepithelial intestinal absorption of P i is mainly

explained by alterations in the apical abundance of Na/

P i -IIb. Indeed, modulation of P i transport through the

cotransporter is regulated by various physiological effec-

tors. Epidermal growth factor, and glucocorticoids inhibit

intestinal sodium-dependent absorption and Na/P i -IIb gene

expression, whereas 1,25(OH) 2 D, estrogen and dietary P i

deprivation stimulate sodium-dependent absorption and

Na/P i -IIb gene expression.

To date numerous studies have shown that the intestinal

absorption of P i is regulated by 1,25(OH) 2 D. The effects of

1,25(OH) 2 D on this process are modulated by the calcitriol-

induced transcription of messenger RNA. Because low

Ba

so

late

ral m

em

bra

ne

Bru

sh

bo

rde

r (lu

min

al) m

em

bra

ne

2Na1 2Na

1

K1 ATPK1

ADP

Na12Na1 2Na1

Na1 Na

1

H1 H1

Na1

2A2

HPO45 HPO4

5

HPO45

HPO45 HPO4

5 HPO45

FIGURE 1 Model of inorganic phosphate (HPO )45

transcellular trans-

port in the proximal convoluted tubule of the mammalian kidney. On the

brush border or luminal membrane, a Na 1 /H 1 exchanger and Na/HPO45

cotransporters operate. (HPO )45

that enters the cell across the luminal

surface mixes with the intracellular metabolic pool of phosphate and is

eventually transported out of the cell across the basolateral membrane via

an anion (A-) exchange mechanism. On the basolateral membrane there

is also a 2 4Na/HPO5 cotransporter and a Na 1 /K 1 -ATPase system. The

ATPase transports the Na 1 out of the cell, maintaining the Na 1 gradient-

driving force for luminal phosphate entry.


dietary intake of P i stimulates the synthesis of calcitriol,

the enhanced P i absorption that occurs concomitantly has

been attributed to the effects of this vitamin D metabolite

on the abundance of the apical Na/P i -IIb protein. Because

in the adult mouse, the calcitriol dependent increase in the

Na/P i -IIb protein is paralleled by an increase in Na/P i -IIb

mRNA, the upregulation of the receptors in response to

this stimulus has been considered secondary to a genomic

mechanism possibly involving the VDR. However, recent

studies ( Capuano et al., 2005 ) indicate that upregulation

of the Na/P i -IIb receptors by a low P i diet occurs normally

in VDR 2 / 2 and 25(OH)D-1 α -hydroxylase-deficient mice,

indicating that the increase in receptors likely involves a

genomic mechanism that does not require VDR and is not

dependent on an increased 1,25(OH) 2 D level. The mecha-

nism for this effect, however, remains unknown.

Although the active transport systems are responsive to

various hormones ( Lee et al. , 1986 ; Rizzoli et al. , 1977 ;

Xu et al., 2001 ), such hormonal provocation plays a rela-

tively minor role in normal phosphate homeostasis ( Takeda

et al., 2004 ). For example, during vitamin D deficiency,

the percentage of P absorbed from the diet is reduced by

only 15%. Moreover, a substantial portion of this decline

is secondary to the failure to absorb calcium, which

results from vitamin D deficiency and the resultant forma-

tion of calcium phosphate that reduces the free phosphate

concentration.

The minimal effects of hormone stimulation on P i

absorption are expected, because the vast majority of

phosphate absorption occurs via the process of diffusional

absorption. This results as a consequence of the relatively

low K m of the active transport process (2 m M ); the luminal

P content during feeding, which generally exceeds 5 m M

throughout the intestine; and the occurrence of net diffu-

sional absorption of P whenever luminal P concentration

exceeds 1.8 m M (a concentration generally exceeded even

when fasting; Karr and Abbott, 1935 ; Walton and Gray,

1979 ; Wilkinson, 1976 ). Given these conditions, the active

component of transport becomes important only under

unusual circumstances, such as when dietary P or vitamin D

is extremely low. Regardless, the bulk of intestinal P

absorption is mediated by a diffusional process, presum-

ably through the paracellular space, and therefore is pri-

marily a function of P intake. Because most diets contain

an abundance of P, the quantity of phosphate absorbed

almost always exceeds the need both under normal circum-

stances and disease states such as uremia. Thus, active,

transcellular P absorption becomes predominant only under

conditions of low luminal P availability, such as dietary P

deprivation and/or excessive luminal P binding ( Lee et al. ,

1979 ; Kurnik and Hruska, 1984 ). Factors that may influ-

ence the diffusional process adversely are the forma-

tion of nonabsorbable calcium, aluminum, or magnesium

phosphate salts in the intestine and age, which reduces P

absorption by as much as 50%.

Renal Excretion of Phosphorus

The kidney is immediately responsive to changes in serum

levels or dietary intake of phosphate. Renal adaptation is

determined by the balance between the rates of glomerular

filtration and tubular reabsorption ( Mizgala and Quamme,

1985 ).

The concentration of phosphate in the glomerular ultra-

filtrate is approximately 90% of that in plasma because not

all of the phosphate in plasma is ultrafilterable ( Harris et al. ,

1977 ). Nondiffusable phosphorus includes plasma P that

is protein bound and a small fraction of plasma P that

complexes with calcium and magnesium. With increasing

serum calcium levels, the calcium–phosphate–protein col-

loid complex increases, reducing the ultrafilterable plasma

P to as little as 75% ( Rasmussen and Tenenhouse, 1995 ).

Because the product of the serum phosphorus concentra-

tion and the glomerular filtration rate (GFR) approximates

the filtered load of phosphate, a change in the GFR may

influence phosphate homeostasis if uncompensated by

commensurate changes in tubular reabsorption.

Normally, 80% to 90% of the filtered phosphate load

is reabsorbed, primarily in proximal tubules, with higher

rates at early segments (S 1 /S 2 versus S 3 ) and in deep neph-

rons ( Agus, 1983 ; Cheng and Jacktor, 1981 ; Dousa and

Kempson, 1982 ; Suki and Rouse, 1996 ). The transcellu-

lar transport of phosphate is a carrier-mediated, saturable

process limited by a transfer maximum or T max . The T max

varies considerably as dietary phosphorus changes, and

the best method to approximate this variable is to measure

maximum phosphate reabsorption per unit volume of glo-

merular filtrate ( T m P/GFR) during acute phosphate infu-

sions. Alternatively, the nomogram developed by Bijvoet

allows estimation of the T m P/GFR with measurement of

phosphate and creatinine excretion and plasma phosphate

concentration ( Walton and Bijvoet, 1975 ).

The major site of phosphate reabsorption is the proxi-

mal convoluted tubule, at which 60% to 70% of reabsorp-

tion occurs ( Fig. 2 ). Along the proximal convoluted tubule

the transport is heterogeneous. In the most proximal por-

tions, the S 1 segment, phosphate reabsorption exceeds that

of sodium and water, whereas more distally, phosphate

reabsorption parallels that of fluid and sodium. Additional

reabsorption in the proximal straight tubule accounts for

15% to 20% of phosphate reclamation. In contrast, there

is little evidence to suggest net P transport in the thin and

thick ascending loops of Henle. However, increasing, but

not conclusive, data support the existence of a P reab-

sorptive mechanism in the distal tubule. Currently, how-

ever, definitive proof for tubular secretion of phosphate in

humans is lacking ( Knox and Haramati, 1981 ).

At all three sites of phosphate reabsorption—the proxi-

mal convoluted tubule, proximal straight tubule, and dis-

tal tubule—several investigators have mapped parathyroid

hormone (PTH)-sensitive adenylate cyclase (see Fig. 2 ;


Knox and Haramati, 1981 ; Morel, 1981 ). Not surpris-

ingly, there is clear evidence that PTH decreases phosphate

reabsorption at these loci by a cAMP-dependent process,

as well as a cAMP-independent signaling mechanism.

Likewise, several investigators have identified FGF-23

receptors in the proximal tubule, the primary site at which

phosphatonins decrease phosphate reabsorption. However,

the colocalization of klotho proteins at these sites is

controversial despite the need for these proteins to acti-

vate the FGF-23 receptors. In fact, the primary localiza-

tion of the klotho proteins is the DCT. Calcitonin-sensitive

adenylate cyclase maps to the medullary and cortical thick

ascending limbs and the distal tubule (see Fig. 2 ; Berndt

and Knox, 1984 ). Nevertheless, calcitonin inhibits phos-

phate reabsorption in the proximal convoluted and proxi-

mal straight tubule, certainly by a cAMP-independent

mechanism that may be mediated by a rise in intracellular

calcium ( Murer et al. , 2000 ). An action of calcitonin on the

distal tubule is uncertain, despite the abundant calcitonin-

sensitive adenylate cyclase.

Hormonal/Metabolic Regulation of Phosphate Transport

Many hormonal and nonhormonal factors regulate renal

reabsorption of P i . The effects of PTH and dietary P i , and

more recently phosphatonins (most notably FGF-23), on

this process have been the subject of detailed investigation.

These studies suggest that NaP i -IIa receptor regulation

depends on its shuttling to/from the brush border mem-

brane of the renal tubule. Thus, reduced P i reabsorption (on

PTH or phosphatonin release) is achieved by downregula-

tion of the receptors at the brush border membranes. Such

downregulation is dependent on endocytic removal of the

receptors from the membranes and subsequent degrada-

tion in lysosomes. As a consequence, subsequent restora-

tion of P i transport upon removal of the hormonal stimulus

depends on de novo synthesis. The detailed mechanism

for recycling of the receptors is provided in a review by

Forster et al. (2006) . In concert with these findings, stud-

ies indicate that expression of the Na/P i -IIa protein at renal

tubular sites is increased in parathyroidectomized rats and

decreased after PTH treatment. In addition, Northern blot

analysis of total RNA shows that the abundance of Na/P i -

IIa-specific mRNA is not changed by parathyroidectomy,

but is decreased minimally in response to the administra-

tion of parathyroid hormone. These data are consistent

with the concept that parathyroid hormone regulation of

renal Na 1 –P i cotransport is determined predominantly by

changes in expression of the Na/P i -IIa protein in the renal

brush border membrane ( Kempson et al. , 1995 ).

In contrast, a requirement for increased P i reabsorption

(in response to a sustained low P i diet) is met by increasing

expression of NaP i -IIa (and NaP i -IIc) at the brush border

membrane. Such upregulation is independent of changes

in transcription or translation. Therefore, increased expres-

sion of the receptors is due to both the stabilization of the

transporter at the membrane and an increased rate of inser-

tion in the membrane. Thus, dietary-induced upregula-

tion is modulated by the presence of scaffolding proteins,

which stabilize action, and the microtubule network, which

facilitates an increased rate of insertion.

Cortex

Outer

medulla

Inner

medulla

Glomerulus

PCT

DCT

CCT

OMCT

IMCT

Phosphatereabsorption

PCT 60–70% 11

PST 15–20% 11

DCT 5–10% 11

CAL 0% 11

0% 2

2

2

11

11

11

11

11

11

2

2

11

11

2

2

2MAL

PTHsensitiveadenylatecyclase

Calcitoninsensitiveadenylatecyclase

PTHinhibited

phosphatereabsorption

Calcitonininhibited

phosphatereabsorption

CAL

MAL

PCT

S1

S2

PSTS3

FIGURE 2 Model of the renal tubule and distribution of phosphate

reabsorption and hormone-dependent adenylate cyclase activity through-

out the structure. The renal tubule consists of a proximal convoluted

tubule (PCT), composed of an S 1 and S 2 segment, a proximal straight

tubule (PST), also known as the S 3 segment, the loop of Henle, the med-

ullary ascending limb (MAL), the cortical ascending limb (CAL), the dis-

tal convoluted tubule (DCT), and three segments of the collecting tubule:

the cortical collecting tubule (CCT), the outer medullary collecting tubule

(OMCT), and the inner medullary collecting tubule (IMCT). Phosphate

reabsorption occurs primarily in the PCT but is maintained in the PST

and DCT as well. In general, parathyroid hormone (PTH) influences

phosphate reabsorption at sites where PTH-dependent adenylate cyclase

is localized. FGF-23 modifies phosphate reabsorption in the PCT, where

receptors for the phosphatonin are located. However, colocalization of the

klotho protein, which is necessary for receptor activation, is controver-

sial, because it is found primarily in the DCT. Calcitonin alters phosphate

transport at sites distinct from those where calcitonin-dependent adenyl-

ate cyclase is present, suggesting that response to this hormone occurs by

a distinctly different mechanism.


Despite these advances in understanding the mecha-

nisms regulating the abundance of Na/P i receptors, there

remains a void in our knowledge regarding the manner in

which the organism maintains overall P i balance. Although

historically PTH is accepted as the most important physi-

ologic influence on renal P excretion and the major deter-

minant of plasma P concentrations through its effect on P i

transport, repeated observations have confirmed that the

balance between urinary excretion and dietary input of P

is maintained not only in normal humans but in patients

with hyper- and hypoparathyroidism. In fact, the renal

tubule has a seemingly intrinsic ability to adjust the reab-

sorption rate of P according to dietary P i intake and the

need and availability of P i to the body ( Levi et al. , 1994 ).

Thus P i reabsorption is increased under conditions of

greater P need, such as rapid growth, pregnancy, lacta-

tion, and dietary restriction. Conversely, in times of sur-

feit, such as slow growth, chronic renal failure, or dietary

excess, renal P reabsorption is curtailed. This adaptive

response is localized in the proximal convoluted tubule

and involves an alteration in the apparent V max of the

high-affinity Na 1 –phosphate cotransport systems. Such

changes in response to chronic changes in P i availability

are characterized by parallel changes in Na 1 –phosphate

cotransport activity, the Na/P i transporter mRNA level,

and protein abundance. In contrast, the acute adaptation to

altered dietary P i is marked by parallel changes in Na 1 –

phosphate cotransporter activity and Na/P i -IIa protein

abundance in the absence of a change in mRNA. Thus, in

response to chronic conditions, protein synthesis is requi-

site in the adaptive response, whereas under acute condi-

tions, the number of Na/P i -IIa cotransporters is changed

rapidly by mechanisms independent of de novo protein

synthesis, such as insertion of existing transporters into

the apical membrane or internalization of existing trans-

porters. Although the signal for the adaptive alteration in

phosphate transport is not yet known, several lines of evi-

dence suggest that renal adaptations to changes in dietary

phosphate occur independently of the known regulators

of renal phosphate transport. In this regard, adaptations to

changes in dietary phosphate mediated by the vitamin D

endocrine system, PTH, and the phosphatonins generally

occur over a period of hours to days and cannot account

for the modulation of renal phosphate excretion that rap-

idly occurs after a phosphate-containing meal. Recently,

Berndt et al. (2007) presented data that support the exis-

tence of an intestinal-renal axis specific for phosphate

that is mediated by an as yet unknown factor, an intestinal

“ phosphatonin. ” These observations suggest that the rapid

renal response to increased dietary phosphate concentra-

tions is due to the regulated production in the duodenum

of a hormone, which increases the renal excretion of phos-

phate. This response dampens large increases in serum

phosphate concentrations that could have a deleterious

effect by enhancing the precipitation of calcium phosphate

salts in soft tissues.

CLINICAL DISORDERS OF PHOSPHATE

HOMEOSTASIS

The variety of diseases, therapeutic agents, and physiologi-

cal states that affect phosphate homeostasis are numerous

and reflect a diverse pathophysiology. Indeed, rational

choice of an appropriate treatment for many of these dis-

orders depends on determining the precise cause for the

abnormality. The remainder of this chapter reviews several

clinical states that represent primary disorders of phos-

phate homeostasis. These include X-linked hypophospha-

temic rickets/osteomalacia (XLH); autosomal-dominant

hypophosphatemic rickets (ADHR); autosomal recessive

hypophosphatemic rickets (ARHR); tumor-induced osteo-

malacia (TIO); hereditary hypophosphatemic rickets with

hypercalciuria (HHRH); Dent’s disease; Fanconi’s syn-

drome (FS), types I and II; and tumoral calcinosis (TC).

Table II documents the full spectrum of diseases in which

disordered phosphate homeostasis occurs. Many of these

are discussed in other chapters.

Impaired Renal Tubular Phosphate

Reabsorption

X-Linked Hypophosphatemic Rickets

X-linked hypophosphatemic rickets/osteomalacia is the

archetypal phosphate-wasting disorder, characterized in

general 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 III ). The clinical

expression of the disease is widely variable even in mem-

bers of the same family, ranging from a mild abnormality,

the apparent isolated occurrence of hypophosphatemia,

to severe bone disease ( Lobaugh et al. , 1984 ). On aver-

age, disease severity is similar in males and females, indi-

cating minimal, if any, gene dosage effect ( Whyte et al. ,

1996 ). The most common clinically evident manifestation

is short stature. This height deficiency is a consequence of

abnormal lower extremity growth, averaging 15% below

normal. In contrast, upper segment growth is not affected.

The majority of children with the disease exhibit enlarge-

ment 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 den-

tin, enthesopathy (calcification of tendons, ligaments, and

joint capsules), and premature cranial synostosis. However,

many of these features may not become apparent until age

6 to 12 months or older ( Harrison et al. , 1966 ). Despite

marked variability in the clinical presentation, bone biop-

sies in affected children and adults invariably reveal osteo-

malacia, the severity of which has no relationship to sex,

the extent of the biochemical abnormalities, or the severity


of the clinical disability. In untreated youths and adults,

serum 25(OH)D levels are normal and the concentra-

tion of 1,25(OH) 2 D is in the low-normal range ( Haddad

et al. , 1973 ; Lyles et al. , 1982 ). The paradoxical occur-

rence of hypophosphatemia and normal serum calcitriol

levels is due to the aberrant regulation of renal 25(OH)D-

1 α -hydroxylase activity as a direct result of abnormal

phosphate transport or of elevated circulating levels of

phosphatonins (e.g., FGF-23), independent of the effects

on phosphate transport. Studies in hyp -mice, the murine

homologue of the human disease, have established that

defective regulation is confined to enzyme localized in the

proximal convoluted tubule, the site of the abnormal phos-

phate transport ( Lobaugh and Drezner, 1983 ; Nesbitt et al. ,

1986, 1987 ; Nesbitt and Drezner, 1990 ).

Pathophysiology

Investigators generally agree that the primary inborn error

in XLH results in an expressed abnormality of the renal

proximal tubule that impairs P i 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. Until recently, whether

this renal abnormality is primary or secondary to the elab-

oration of a humoral factor has been controversial. In this

regard, demonstration that renal tubule cells from hyp -

mice maintained in primary culture exhibit a persistent

defect in renal P i transport ( Bell et al. , 1988 ; Dobre et al. ,

1990 ), likely due to decreased expression of the Na 1 –

phosphate cotransporter mRNA and immunoreactive pro-

tein ( Tenenhouse et al. , 1994, 1995 ; Collins and Ghishan,

1994 ), supported the presence of a primary renal abnor-

mality. In contrast, transfer of the defect in renal P i trans-

port to normal and/or parathyroidectomized normal mice

parabiosed to hyp -mice implicated a humoral factor in the

pathogenesis of the disease ( Meyer et al. , 1989a, 1989b ).

Subsequent studies, however, have provided compelling

evidence that the defect in renal P i transport in XLH is

secondary to the effects of a circulating hormone or met-

abolic factor. Thus, immortalized cell cultures from the

renal tubules of hyp -mice exhibit normal Na 1 –phosphate

transport, suggesting that the paradoxical effects observed

in primary cultures may represent the effects of impressed

memory and not an intrinsic abnormality ( Nesbitt et al. ,

1995, 1996 ). Moreover, the report that cross-transplantation

of kidneys in normal and hyp -mice results in neither trans-

fer of the mutant phenotype nor its correction unequivo-

cally established the humoral basis for XLH ( Nesbitt et al. ,

1992 ). Subsequent efforts, which resulted in localization

of the gene encoding the primary renal Na 1 –phosphate

cotransporter to chromosome 5, further substantiated the

conclusion that the renal defect in brush border membrane

phosphate transport is not intrinsic to the kidney ( Kos

et al. , 1994 ). Although these data establish the presence of

a humoral abnormality in XLH, the identity of the putative

TABLE II Diseases of Disordered Phosphate

Homeostasis

Increased phosphate

Reduced renal phosphate excretion

Renal failure

Hypoparathyroidism

Tumoral calcinosis a

Hyperthyroidism

Acromegaly

Diphosphonate therapy

Increased phosphate load

Vitamin D intoxication

Rhabdomyolysis

Cytotoxic therapy

Malignant hyperthermia

Decreased phosphate

Decreased gastrointestinal absorption

Phosphate deprivation

Gastrointestinal malabsorption

Increased renal phosphate excretion

Hyperparathyroidism

X-linked hypophosphatemic rickets/osteomalacia a

Fanconi’s syndrome, type I a

Familial idiopathic

Cystinosis (Lignac–Fanconi disease)

Hereditary fructose intolerance

Tyrosinemia

Galactosemia

Glycogen storage disease

Wilson’s disease

Lowe’s syndrome

Fanconi’s syndrome, type II a

Vitamin D–dependent rickets

Autosomal-dominant hypophosphatemic rickets a

Autosomal-recessive hypophosphatemic rickets a

Dent’s disease (X-linked recessive hypophosphatemic

rickets) a

Tumor-induced osteomalacia a

Hereditary hypophosphatemic rickets with hypercalciuria a

Transcellular shift

Alkalosis

Glucose administration

Combined mechanisms

Alcoholism

Burns

Nutritional recovery syndrome

Diabetic ketoacidosis

a Primary disturbance of phosphate homeostasis.


TABLE III Biochemical and Genetic Characteristics of Primary Disturbances of Phosphate Homeostasisa

XLH HHRH ADHR ARHR Dent’s

Disease

TIO FS I FS II TC

Calcium metabolism

Serum Ca N/LN N/HN N/LN N/LN N N/LN N/LN N/HN N/HN

Urine Ca ↓ ↑ ↓ ↓ ↑ ↓ ↓ ↑ ↑

Serum PTH N N/LN N N N/LN N N N/LN N

Phosphate metabolism

Serum P ↓ ↓ ↓ ↓ N/ ↓ ↓ ↓ ↓ ↑

T m P/GFR ↓ ↓ ↓ ↓ N/ ↓ ↓ ↓ ↓ ↑

GI function

P absorption ↓ ↑ ↓ ↓ ? ↓ ↓ ↑ ↑

Ca absorption ↓ ↑ ↓ ↓ ↑ ↓ ↓ ↑ ↑

Serum biochemistries

Alkaline

phosphatase

N/ ↑ N/ ↑ N/ ↑ N/ ↑ N/ ↑ N/ ↑ N/ ↑ N/ ↑ N

Vitamin D metabolism

Serum

25(OH)D

N N N N N N N N N

Serum 1,25(OH) 2 D ( ↓ ) ↑ ( ↓ ) ( ↓ ) ↑ ↓ ( ↓ ) ↑ ↑

Gene(s)

mutation

PHEX Na/Pi-IIC FGF-23 DMP1 CLCN5 – – – FGF-23,

GLANT3

KLOTHO

a Modifi ed from Econs et al. (1992). XLH, X-linked hypophosphatemic rickets; HHRH, hereditary hypophosphatemic rickets with hypercalciuria; ADHR, autosomal-dominant

hypophosphatemic rickets; ARHR, autosomal recessive hypophosphatemic rickets; TIO, tumor-induced osteomalacia; FS I, Fanconi’s syndrome type I; FS II, Fanconi’s syndrome type

II; TC, tumoral calcinosis. N, normal; LN, low normal; HN, high normal; ↑ , increased; ↓ , decreased; ( ↓ ), decreased relative to the serum phosphorus concentration.

factor, the spectrum of its activity, and the cells producing

it have not been definitively elucidated until recently.

Genetic Defect

Efforts to better understand XLH have more recently

included attempts to identify with certainty the genetic

defect underlying this disease. In 1986, Read and co-

workers and Machler and colleagues reported link-

age of the DNA probes DXS41 and DXS43, which had

been previously mapped to Xp22.31–p21.3, to the HYP

gene locus. In subsequent studies, Thakker et al. (1987)

and Albersten et al. (1987) 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 determina-

tion of the genetic map along the Xp22–p21 region of the

X chromosome and only allowed identification of flank-

ing markers for the HYP locus 20 cM apart. However, the

HYP consortium (HYP Consortium, 1995), in a study of

some 20 multigenerational pedigrees, used a positional

cloning approach to refine mapping of the Xp22.1–p21

region of the X chromosome, identify tightly linked flank-

ing markers for the HYP locus, construct a YAC contig

spanning the HYP gene region, and eventually clone and

identify the disease gene as PHEX , a phosphate-regulat-

ing gene with homologies to endopeptidases located on the

X chromosome. The PHEX locus was mapped to Xp22.1

and more than 140 loss-of-function PHEX mutations (see

http://www.envbiocourse.rutgers.edu/eu-us/pages/extras/

software/DatabasesListNAR2002/summary/145.html ) have

been reported to date in patients with XLH ( Holm et al.,

1997 ). However, no genotype/phenotype correlations have

been recognized, albeit changes in conserved amino acids

more often result in clinical disease than changes in non-

conserved amino acids ( Filisetti et al. , 1999 ). Further stud-

ies documented that the murine homologue of the human


disease, the hyp- mouse, has a phenotype identical to that

evident in patients with XLH and is due to a large dele-

tion of the 3 ’ region of the Phex gene ( Beck et al., 1997 ).

Collectively, these findings indicate that a mutation in the

PHEX/Phex gene is responsible for the phenotypic changes

in patients with XLH and the hyp- mouse.

The PHEX gene has 22 exons and encodes for a 749-

amino-acid glycoprotein, which has homology with

members of the M13 membrane-bound zinc metalloendo-

peptidase family. Investigation of murine tissues and cell

cultures revealed that Phex is predominantly expressed

in bones and teeth ( Du et al., 1996 ; Ruchon et al., 1998 ),

although detectable Phex mRNA and/or protein have been

found in lung, brain, muscle gonads, skin and parathy-

roid glands. The PHEX/Phex expression in bone is lim-

ited to cells of the osteoblast lineage ( Ruchon et al., 1998 ;

Thompson et al., 2002 ), osteoblasts and osteocytes.

The physiological function of the PHEX/Phex gene prod-

uct and the mechanisms that lead to the biochemical and skel-

etal abnormalities evident in patients with XLH and in the

hyp -mouse remain ill-defined. Because PHEX/Phex codes for

a membrane-bound enzyme, several groups have postulated

that PHEX/Phex activates or degrades a putative phosphate-

and bone mineralization-regulating factor(s), “ phosphato-

nin ” or “ minhibin, ” which are involved in the regulation of

phosphate and mineralization processes (Rasmussen and

Tenenhouse 1995; Nesbitt et al., 1992 ; Ecarot et al., 1992 ;

Econs and Drezner 1994; Bowe et al., 2001 ). Studies in

patients with XLH and the hyp -mouse, as well as in a wide

variety of other P i wasting disorders, have identified several

candidate proteins as the phosphatonin(s) or minhibin(s),

including FGF-23 (Fukomoto and Yamashita 2002; Liu et al.,

2006 ), sFRP4 ( Kumar, 2002 ; Berndt et al., 2003 ), MEPE

( Argiro et al., 2001 ; Rowe et al. , 2004 ) and FGF-7 ( Carpenter

et al., 2005 ). Because abnormal production or circulat-

ing levels of several of these factors have been identified in

patients with XLH and in hyp -mice, until recently it remained

unknown whether a single phosphatonin is responsible for the

physiological abnormalities of the disease or a family of pro-

teins works cooperatively to create the HYP phenotype.

Progress in this regard has been limited by unsuccess-

ful efforts to rescue the HYP phenotype and determine

the physiologically relevant site for the PHEX mutation.

In attempts to determine whether abnormal PHEX/Phex

expression in bone cells alone is the determining abnor-

mality underlying the pathogenesis of XLH, several

investigators have used osteoblast/osteocyte targeted over-

expression of Phex in order to normalize osteoblast min-

eralization in vitro and rescue the HYP phenotype in vivo

( Liu et al., 2002 ; Bai et al., 2002 ). Surprisingly, however,

these studies documented that restoration of Phex expres-

sion 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. (2002) and

Bai et al. (2002) found that transgenic hyp -mice main-

tained characteristic hypophosphatemia and abnormal

vitamin D metabolism, as well as histological evidence of

osteomalacia, despite expressing abundant Phex mRNA

and enzyme activity in mature osteoblasts and osteocytes,

under the control of the bone-specific promoters osteocal-

cin and ColA1 (2.3 kb). These findings suggest that expres-

sion of Phex at sites other than bone is responsible for the

Hyp phenotype. However, similar studies performed with

transgenic mice in which Phex overexpression was under

the regulation of the ubiquitous human β -actin promoter

likewise failed to normalize the phosphate homeostasis

( Erben et al., 2005 ).

In recent studies, however, Yuan et al. (2007) attempted

to explore this apparent paradox by evaluating whether

conditional inactivation of Phex in osteoblasts and osteo-

cytes generates a biochemical phenotype similar to that in

mice with a global Phex knockout and comparable to that

in hyp- mice. To accomplish this goal they generated mouse

lines with global and targeted deletion of exon 17 from the

Phex gene, which codes a portion of the protein crucial for

bioactivity. Their studies established unequivocally that the

mutation of the Phex gene in osteoblasts and osteocytes

alone is sufficient to generate the classical HYP phenotype.

Indeed, the targeted knockout mice exhibit a biochemi-

cal phenotype (phosphorus homeostatic abnormalities and

aberrant vitamin D metabolism) no different than that of

the global knockout mice and indistinguishable from that

in the hyp- mouse. Moreover, investigation of the bone

pathology in the targeted knockout mice further established

that mutation of the Phex gene in the osteoblasts and osteo-

cytes is a sufficient abnormality to produce the HYP phe-

notype. Hence, bone biopsies from the targeted knockout

mice displayed histomorphological evidence of osteomala-

cia and cannicular disorganization indistinguishable from

those observed in bone biopsies from the global knockout

mice, as well as those from hyp- mice.

Although these observations provide compelling evidence

that aberrant PHEX / Phex function in osteoblasts and osteo-

cytes alone underlies the characteristic biochemical and path-

ological phenotype in hyp- mice and likely XLH, a surprising

discordance between the biochemical phenotype in the tar-

geted knockout mice from that in both the global knockout

and hyp- mice became apparent with further study. Previous

investigations have implicated the phosphatonins as critical

factors in the pathogenesis of XLH and have identified that

the production rate and/or the circulating level of FGF-23,

MEPE, and sFRP-4, are increased in affected humans and/or

the hyp- mouse. However, Yuan et al. (2007) found that the

targeted knockout mice had only an increased osseous pro-

duction rate and serum level of FGF-23, whereas the global

knockout and hyp- mice exhibited increased production and

serum levels of FGF-23, MEPE, and sFRP-4. These observa-

tions provide the first successful attempt to discern whether


integrated effects of these hormones or the activity of a single

phosphatonin is essential for expression of the disease phe-

notype in hyp -mice and affected patients with XLH. Indeed,

the 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 FGF-23 concentration appear requisite

for this biological function. Therefore, FGF-23 is likely the

phosphatonin pivotal to the pathogenesis of XLH, and the

role of MEPE and sFRP-4, if any, remains uncertain.

Pathogenesis

Despite the remarkable advances that have been made in

understanding the genetic abnormality and pathophysi-

ology of XLH, the detailed pathogenetic mechanism

underlying this disease remains unknown. Nevertheless,

several observations suggest the likely cascade of events

that result in the primary abnormalities characteristic

of the syndrome. In this regard, the X-linked dominant

expression of the disorder with little, if any, gene dos-

age effect likely results from PHEX mutations that result

in a haploinsufficiency defect, in which one half the nor-

mal gene product (or null amounts) causes the phenotype.

The alternative possibility that the PHEX gene results in a

dominant-negative effect is unlikely because, inconsistent

with this prospect, several mutations reported in affected

humans ( Francis et al. , 1997 ) and the murine Gy mutation

almost certainly result in the lack of message production

( Meyer et al. , 1998 ). In any case, it is tempting to specu-

late that the PHEX gene product acts directly or indirectly

on a phosphaturic factor (e.g., FGF-23) that regulates renal

phosphate handling. Given available data, the PHEX gene

product, a putative cell membrane-bound enzyme, may

function normally to inactivate “ phosphatonin, ” a phospha-

turic hormone. However, data from parabiotic studies of

normal and hyp -mice argue strongly that extracellular deg-

radation of the phosphaturic factor does not occur. Indeed,

such activity would preclude transfer of the hyp -mouse

phenotype to parabiosed normals. Alternatively, the PHEX

gene product may function intracellularly to regulate phos-

phatonin production. In this regard, Jalal et al. (1991)

reported the internalization of neutral endopeptidase and

a potential role for this enzyme in intracellular metabo-

lism. In addition, Thompson et al. (2002) reported that the

PHEX protein in osteoblasts is found predominantly in the

Golgi apparatus and the endoplasmic reticulum.

In any of these cases, a defect in the PHEX gene will

result in overproduction and circulation of phosphatonin

and consequent decreased expression of the renal Na 1 –

phosphate cotransporter, the likely scenario in the patho-

genesis of XLH. The possible mechanism by which PHEX

regulates the production of FGF-23, the requisite phospha-

tonin for the expression of the HYP phenotype, was recently

discovered by Yuan and Drezner (unpublished observa-

tions). Their studies indicate that the loss of function

Phex mutation in hyp -mice results in decreased production

of 7B2, an intracellular chaperone protein, which proteo-

lytically activates subtilin-like prohormone convertase 2

(SPC2), enabling cleavage of hormones to inactive pep-

tides and prohormones to active hormones. The decreased

activity of the 7B2–SPC2 enzyme complex decreases FGF-

23 degradation in the osteoblast, increasing the concentra-

tion of the intact active FGF-23. In addition, the decreased

7B2–SPC2 activity limits conversion of DMP1 (104 kDa)

to active DMP1 (57 kDa), the relative absence of which

increases FGF-23 mRNA and consequent protein produc-

tion. These concordant effects on FGF-23 degradation and

production result in the increased circulating FGF-23 and

production of the HYP phenotype.

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 vita-

min D intoxication and renal damage. Current treatment

strategies for children directly address the combined cal-

citriol and phosphorus deficiency characteristic of the dis-

ease. Generally, the regimen includes a period of titration to

achieve a maximum dose of calcitriol, 40 to 60 ng/kg/day in

two divided doses, and phosphorus, 1 to 2 g/day in four to

five divided doses (Friedman and Drezner 1991, 1993). Such

combined therapy often improves growth velocity, normal-

izes lower extremity deformities, and induces healing of the

attendant bone disease. Of course treatment involves a sig-

nificant risk of toxicity that is generally expressed as abnor-

malities of calcium homeostasis and/or detrimental effects on

renal function secondary to abnormalities such as nephrocal-

cinosis. In addition, refractoriness to the growth-promoting

effects of treatment is often encountered, particularly in

youths presenting at below the 5th percentile in height

( Friedman et al. , 1993 ). Several studies, however, indicate

that the addition of growth hormone to conventional therapy

increases growth velocity significantly. Unfortunately, such

a benefit is realized more frequently in younger patients, and

disproportionate growth of the trunk often continues to man-

ifest. Moreover, the definitive impact of growth hormone

treatment on adult height remains unknown ( Wilson, 2000 ).

More recently, several investigators have suggested that

the effects of combination therapy with calcitriol and phos-

phate may be enhanced with the concurrent use of cina-

calcet, a calcimimetic agent, which reduces parathyroid

hormone. Use of cinacalcet decreases the elevated parathy-

roid hormone observed occasionally in untreated patients

and frequently in treated patients, limiting the phosphaturic

effects of PTH and permitting more optimal restoration to

normal of the renal TmP/GFR and serum phosphorus con-

centration in treated patients.

Therapy in adults is reserved for episodes of intractable

bone pain and refractory nonunion bone fractures.


Hereditary Hypophosphatemic Rickets with Hypercalciuria

This rare genetic disease is characterized by hypophos-

phatemic rickets with hypercalciuria ( Tieder et al. , 1985 ).

The cardinal biochemical features of the disorder include

hypophosphatemia due to increased renal phosphate clear-

ance and normocalcemia. In contrast to other diseases in

which renal phosphate transport is limited, patients with

HHRH exhibit increased 1,25(OH) 2 D production (see

Table III ). The resultant elevated serum calcitriol levels

enhance gastrointestinal calcium absorption, which in turn

increases the filtered renal calcium load and inhibits para-

thyroid secretion ( Tieder et al. , 1985 ). These events cause

the hypercalciuria observed in affected patients.

The clinical expression of the disease is heterogeneous,

although initial symptoms, evident at 6 months to 7 years

of age, generally consist of bone pain and/or deformities

of the lower extremities. The bone deformities vary from

genu varum or genu valgum to anterior external bowing of

the femur and coxa vara. Additional features of the disease

include short stature, muscle weakness, and radiographic

signs of rickets or osteopenia. These various symptoms

and signs may exist separately or in combination and may

be present in a mild or severe form. Relatives of patients

with evident HHRH may exhibit an additional mode of

disease expression. These subjects manifest hypercalci-

uria and hypophosphatemia, but the abnormalities are less

marked and occur in the absence of discernible bone dis-

ease ( Tieder et al. , 1987 ). Bone biopsies in children with

characteristic HHRH exhibit classical osteomalacia, but

the mineralization defect appears to vary in severity with

the magnitude of the hypophosphatemia. Histological mea-

surements are within the normal range in family members

with idiopathic hypercalciuria.

Pathophysiology and Genetics

Liberman and co-workers ( Tieder et al. , 1985, 1987

Liberman, 1988 ) have presented data that indicate the primary

inborn error underlying this disorder is an expressed abnor-

mality in the renal proximal tubule, which impairs phosphate

reabsorption. They propose that this pivotal defect results in

enhanced renal 25(OH)D-1 α -hydroxylase, thus promoting

the production of 1,25(OH) 2 D and increasing its serum lev-

els. Consequently, intestinal calcium absorption is augmented,

resulting in the suppression of parathyroid function and an

increase of the renal filtered calcium load. The concomitant

prolonged hypophosphatemia diminishes osteoid mineraliza-

tion and accounts for the ensuing rickets and/or osteomalacia.

The suggestion that abnormal phosphate transport

results in increased calcitriol production remains untested.

Indeed, the elevation of 1,25(OH) 2 D in patients with

HHRH is a unique phenotypic manifestation of the dis-

ease 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 uniformly impairs phosphate transport

but not 25(OH)D-1 α -hydroxylase activity. Alternatively, the

aberrant regulation of vitamin D metabolism in other hypo-

phosphatemic disorders may occur independently (e.g., in

XLH secondary to the PHEX gene abnormality or FGF-23)

and override the effects of the renal phosphate transport.

Extensive studies in a large consanguineous Bedouin

kindred have established that HHRH is transmitted in an

autosomal recessive inheritance pattern. Under such circum-

stances, the variability in this disorder may be explained by

assuming that individuals with HHRH or idiopathic hyper-

calciuria are homozygous and heterozygous, respectively, for

the same mutant allele. Early studies focused on Na/P i -IIa

as a candidate gene underlying HHRH. Indeed, Beck et al.

(1998) reported that homozygous ablation of this murine

gene ( SLC34a1 ) leads to hypophosphatemia, renal phos-

phate wasting, increased serum 1,25(OH) 2 D levels, and

hypercalciuria. These findings were evident at weaning,

but the magnitude of the changes decreased with increas-

ing age. Furthermore, Na/P i -IIa-ablated mice lack the typi-

cal features of rickets ( Beck et al., 1998 ; Gupta et al., 2001 ;

Tenenhouse, 2005 ). Therefore, it was not surprising that

SLC34A1 mutations were excluded in genomic DNA from

affected members of several kindred with HHRH ( Jones

et al., 2001 ; van den Heuvel et al., 2001 ). More recently,

Bergwitz et al. (2006) performed a genome-wide linkage

scan, combined with a homozygosity mapping approach

in an extended Bedouin kindred to map the genetic defect

responsible for HHRH to a 1.6-Mbp interval in chro-

mosome region 9q34. Na/P i -IIc ( SLC34A3 ), a plausible

candidate gene in this region, was sequenced and all indi-

viduals with HHRH were homozygous for a 1-nt deletion,

c 228delC. This mutation produces a nonfunctional Na/P i -

IIc protein that is truncated within the first membrane-span-

ning domain. Consistent with these observations, several

compound heterozygous missense mutations or deletions

were found in affected individuals from three additional

unrelated kindreds, which were not present in healthy con-

trols. Moreover, several investigators ( Ichikawa et al., 2006 ;

Lorenz-Depiereux et al., 2006 ) have confirmed mutations

in SLC34A3 in multiple additional families.

Although the function of the NaPi-IIc receptor in the

regulation of renal phosphate transport was considered

minimal, the identification of a mutation in the SLC34A3

gene as the cause of HHRH has brought new perspective

to the role of this receptor. Indeed, a recent series of stud-

ies (Tenenouse et al., 2003; Miyamoto et al., 2004 ) provide

undeniable evidence that NaP i -IIC appears to be critically

involved in regulating phosphate homeostasis.

Treatment

In accord with the hypothesis that a singular defect

in renal phosphate transport underlies HHRH, affected


patients have been treated successfully with high-dose

phosphorus (1–2.5 g/day in five divided doses) alone. In

response to therapy, bone pain disappears and muscular

strength improves substantially. Moreover, the majority

of treated subjects exhibit accelerated linear growth and

radiologic signs of rickets disappear completely within 4 to

9 months. Concordantly, serum phosphorus values increase

toward normal, the 1,25(OH) 2 D concentration decreases,

and alkaline phosphatase activity declines. Despite this

favorable response, limited studies indicate that such treat-

ment does not heal the associated osteomalacia. Therefore,

further investigation is necessary to determine whether

phosphorus alone is truly sufficient for this disorder.

Autosomal-Dominant Hypophosphatemic Rickets

Several studies have documented an autosomal-dominant

inheritance, with incomplete penetrance, of a hypophos-

phatemic disorder similar to XLH ( Harrison and Harrison,

1979 ). The phenotypic manifestations of this disorder

include lower extremity deformities and rickets/osteoma-

lacia. Indeed, affected patients display biochemical and

radiographic abnormalities indistinguishable from those of

individuals with XLH. These include hypophosphatemia

secondary to renal phosphate wasting and normal levels of

parathyroid hormone and 25(OH)D, as well as inappropri-

ately normal (relative to the serum phosphorus concentra-

tion) 1,25(OH) 2 D (see Table III ). However, unlike patients

with XLH, some with ADHR display variable incomplete

penetrance and delayed onset of penetrance (Econs and

McEnery, 1997). Thus, long-term studies indicate that a few

of the affected female patients exhibit delayed penetrance

of clinically apparent disease and an increased tendency for

bone fracture, uncommon occurrences in XLH. Moreover,

these individuals present in the second through the fourth

decade with weakness and bone pain but do not have lower

extremity deformities. Further, other patients with the dis-

order present during childhood with phosphate wasting,

rickets, and lower extremity deformity but manifest postpu-

bertal loss of the phosphate-wasting defect. Finally, a few

apparently unaffected individuals have been identified, who

seemingly are carriers for the ADHR mutation.

An apparent forme fruste of this disease, (autosomal-

dominant) hypophosphatemic bone disease, has many of

the characteristics of XLH and ADHR, but reports indicate

that affected children display no evidence of rachitic dis-

ease ( Scriver et al. , 1977, 1981 ). Because this syndrome is

described in only a few small kindreds and radiographically

evident rickets is not universal in children with familial

hypophosphatemia, these families may have ADHR. Further

observations are necessary to discriminate this possibility.

Pathophysiology and Genetics

The primary inborn error in ADHR results in an expressed

abnormality of the renal proximal tubule that impairs P i

reabsorption. Until recently, whether this renal abnormality is

primary or secondary to the elaboration of a humoral factor

has been controversial. However, identification of the genetic

defect underlying this disease has established that hormonal

dysregulation is the pivotal abnormality in this disorder. In this

regard, studies localized the ADHR gene to a 6.5-cM interval

on chromosome 12p13 ( Econs et al. , 1997 ). Moreover,

extending these studies, the ADHR Consortium (2000) used

a positional cloning approach to identify 37 genes within

4 Mb of the genomic sequence in the 6.5-cM interval and

identified missense mutations in a gene encoding FGF-23.

Three different mutations were found in four kindreds,

each resulting in amino acid substitutions in the FGF-23

at R176Q, R179Q, and R179W. These mutations occur at

a protease cleavage site (RXXR) and the resulting mutant

FGF-23 molecules are resistant to cleavage ( White et al.,

2001a, 2001b ; Shimada et al., 2002 ; Bai et al., 2003 ). The

impact of these mutations is the inability to degrade the FGF-

23, a relevant abnormality because only the full-length mol-

ecule has biological effects in vivo ( Shimada et al., 2002 ).

Hence the resulting abundance of full-length FGF-23 results in

the hypophosphatemia and attendant rickets and osteomalacia

characteristic of the disorder. The relationship of the elevated

FGF-23 concentration to active disease has recently been inves-

tigated by Imel et al. (2007) . They discovered that FGF-23

concentrations are not universally elevated in ADHR. Rather,

the ADHR symptoms and disease severity likely fluctuate with

the FGF-23 concentration and the ability to alter the FGF-23

concentration changes the clinical phenotype, resulting in the

observed delayed penetrance, and also in at least temporary

resolution of the phenotype in some individuals. Such vari-

ability likely involves an intrinsic but unknown physiological

alteration in the metabolism of FGF-23 leading to resolution of

ADHR features.

Treatment

Although studies of treatment in patients with ADHR are

not available, the pathogenetic role of FGF-23 in the devel-

opment of the disease suggests that therapy effective in

patients with XLH will likewise benefit those with ADHR.

Hence, treatment with calcitriol and phosphorus supplemen-

tation has been employed in a limited number of the affected

patients. Evaluation of treatment outcome will likely reveal

only partial success, similar to that in patients with XLH.

Autosomal Recessive Hypophosphatemic Rickets

Recent studies have identified ARHR in two unrelated,

consanguineous kindreds as a genetically transmitted form

of hypophosphatemic rickets. Affected individuals present

with renal phosphate wasting, rachitic changes, and lower

limb deformity. In family 1 there were three affected sis-

ters and in family 2 a single affected female. The parents

and siblings of these individuals did not exhibit any clini-

cal or biochemical evidence of the disease.


At presentation members of family 1 in mid- to late

infancy had radiographic evidence of rickets or clinically

evident rickets with progressive lower limb deformity. In

family 2 the affected individual presented at 8 years of

age with a mild genu valgum. Biochemical abnormalities

at baseline included hypophosphatemia, renal phosphate

wasting, normal parathyroid function, normocalcemia,

normal urinary calcium, and elevated alkaline phospha-

tase. Serum 1,25(OH) 2 D levels were inappropriately nor-

mal in the presence of hypophosphatemia (see Table III ).

Bone biopsy in an affected individual confirmed severe

osteomalacia, marked by excess osteoid and extended

mineralization lag time, as well as a disrupted osteocyte

lacunocanalicular system. Serum FGF-23 levels were

overtly elevated or overlapped the upper normal range.

Biochemical studies obtained during adulthood showed

persistent renal phosphate wasting, and consequent hypo-

phosphatemia and linear growth was heterogeneous.

Testing for FGF-23 and PHEX mutations was negative.

Studies of the Dmp1 -null mouse provided insight to

the genetic defect underlying ARHR. Dmp1 is a member

of the SIBLING family (Fisher and Fedarko 2003) that is

primarily expressed in mineralized tissues, most notably

osteocytes ( Toyosawa et al., 2001 ). Absence of this pro-

tein in null mice results in mild hypocalcemia and severe

hypophosphatemia secondary to increased renal phosphate

clearance. The enhanced renal phosphate clearance is due

to an elevated serum FGF-23 concentration, which is asso-

ciated with increased FGF-23 mRNA expression in bone.

Moreover, newborn mice lacking Dmp1 develop radio-

logical evidence of rickets and an osteomalacia phenotype

with age ( Ling et al., 2005 ). These observations highlight

that genetic removal of Dmp1 from the skeletal matrix in

mice leads to altered skeletal mineralization and disturbed

phosphate homeostasis associated with increased FGF-23

production, abnormalities similar to those observed in indi-

viduals with ARHR.

Thus, Feng et al. (2006) undertook a candidate gene

approach using direct sequence analyses to test ARHR

families for a mutation in DMP1 . In the affected individ-

uals in family 1, they detected a homozygous deletion of

nucleotides 1484–1490 (1484–1490del) in DMP1 exon 6,

resulting in a frameshift that replaced the conserved C-

terminal 18 residues with 33 unrelated residues. The

affected individual in family 2 had a biallelic nucleotide

substitution in the DMP1 start codon (ATG to GTG, or

A1 → G) and a consequent substitution of the initial methi-

onine with valine (M1V), resulting in predicted loss of the

highly conserved 16-residue DMP1 signal sequence. These

mutations segregated with the disorder in both kindreds,

and neither DMP1 mutation was found in 206 control

alleles. Mutational analyses of an additional 19 hypo-

phosphatemic individuals negative for PHEX and FGF-23

mutations, with no known history of consanguinity, did not

uncover any disease-causing changes in DMP1.

Treatment

Studies in the Dmp1 -null mice revealed that restoring

serum phosphate to normal level by feeding a high-phos-

phate diet rescues the radiological appearance of rick-

ets owing to correction of the mineralization defect at

the growth plate. However, although the osteomalacia

improved with a high-phosphate diet, the bone pheno-

type was not completely rescued, consistent with similar

observations in hyp -mice. Thus, the treatment strategy for

ARHR will likely include use of high-dose phosphate sup-

plements and calcitriol, albeit complete bone healing is not

anticipated.

Tumor-Induced Osteomalacia

Since 1947 there have been reports of well more than 200

patients in whom rickets and/or osteomalacia has been

induced by various types of tumors. In at least half of the

reported cases a tumor has been clearly documented as

causing the rickets/osteomalacia, because the metabolic

disturbances improved or disappeared completely on

removal of the tumor. In the remainder of cases, patients

had inoperable lesions, and investigators could not deter-

mine the effects of tumor removal on the syndrome or sur-

gery did not result in complete resolution of the evident

abnormalities during the period of observation.

Affected patients generally present with bone and

muscle pain, muscle weakness, rickets/osteomalacia, and

occasionally recurrent fractures of long bones. Additional

symptoms common to younger patients are fatigue, gait

disturbances, slow growth, and bowing of the lower

extremities. Biochemistries include hypophosphatemia sec-

ondary to renal phosphate wasting and normal serum lev-

els of calcium and 25(OH)D. Serum 1,25(OH) 2 D is overtly

low or low relative to the prevailing hypophosphatemia (see

Table III ). Aminoaciduria, most frequently glycinuria, and

glucosuria are occasionally present. Radiographic abnor-

malities include generalized osteopenia, pseudofractures,

and coarsened trabeculae, as well as widened epiphyseal

plates in children. The histologic appearance of trabecular

bone in affected subjects most often reflects the presence

of low-turnover osteomalacia. In contrast, bone biopsies

from the few patients who have tumors that secrete a non-

parathyroid hormone factor(s), which activates adenylate

cyclase, exhibit changes consistent with enhanced bone

turnover, including an increase in osteoclast and osteoblast

number.

The large majority of patients with this syndrome har-

bor tumors of mesenchymal origin and include primitive-

appearing, mixed connective tissue lesions, osteoblastomas,

nonossifying fibromas, and ossifying fibromas. However,

the frequent occurrence of Looser zones in the radiographs

of moribund patients with carcinomas of epidermal and

endodermal derivation indicates that the disease may be sec-

ondary to a variety of tumor types. Indeed, the observation


of tumor-induced osteomalacia concurrent with breast

carcinoma ( Dent and Gertner, 1976 ), prostate carcinoma

( Lyles et al. , 1980 ; Murphy et al. , 1985 ; Hosking et al. ,

1975 ), oat-cell carcinoma ( Leehey et al. , 1985 ), small-cell

carcinoma ( Shaker et al. , 1995 ), multiple myeloma, and

chronic lymphocytic leukemia ( McClure and Smith, 1987 )

supports this conclusion. In addition, the occurrence of

osteomalacia in patients with widespread fibrous dysplasia

of bone ( Dent and Gertner, 1976 ; Saville et al. , 1995 ), neu-

rofibromatosis ( Weidner and Cruz, 1987 ; Konishi et al. ,

1991 ), and linear nevus sebaceous syndrome ( Carey et al. ,

1986 ) could also be tumor induced. Although proof of a

causal relationship in these disorders has been precluded in

general by an inability to excise the multiplicity of lesions

surgically, in one case of fibrous dysplasia, removal of vir-

tually all of the abnormal dysplastic lesions did result in

appropriate biochemical and radiographic improvement.

Regardless of the tumor cell type, the lesions at fault

for the syndrome are often small and difficult to locate and

present in obscure areas, which include the nasopharynx, a

sinus, the popliteal region, and the suprapatellar area. In any

case, a careful and thorough examination is necessary to

document or exclude the presence of such a tumor. Indeed,

attempts at PET/CT and/or MRI localization of the tumor

are often required. In addition, several groups have used

octreotide scanning and other forms of nucleotide scintigra-

phy to identify suspected, but nonlocalized, tumors.

Pathophysiology

The relatively infrequent occurrence of this disorder has

confounded attempts to determine the pathophysiological

basis for TIO. Nevertheless, most investigators agree that

tumor production of a humoral factor(s) that may affect

multiple functions of the proximal renal tubule, particularly

phosphate reabsorption, is the probable pathogenesis of the

syndrome. This possibility is supported by (1) the presence

of phosphaturic activity in tumor extracts from three of

four patients with TIO ( Aschinberg et al. , 1977 ; Yoshikawa

et al. , 1977 ; Lau et al. , 1979 ); (2) the absence of parathyroid

hormone and calcitonin from these extracts and the apparent

cyclic AMP-independent action of the extracts; (3) the occur-

rence of hypophosphatemia and increased urinary phosphate

excretion in heterotransplanted tumor-bearing athymic nude

mice ( Miyauchi et al. , 1988 ); (4) the demonstration that

extracts of the hetero-transplanted tumor inhibit renal 25-

hydroxyvitamin D-1 α -hydroxylase activity in cultured kid-

ney cells ( Miyauchi et al. , 1988 ); and (5) the coincidence of

aminoaciduria and glycosuria with renal phosphate wasting

in some affected subjects, indicative of complex alterations

in proximal renal tubular function ( Drezner and Feinglos,

1977 ). Indeed, partial purification of “ phosphatonin ” from

a cell culture derived from a hemangioscleroma causing

tumor-induced osteomalacia has reaffirmed this possibility

( Cai et al. , 1994 ). These studies revealed that the putative

phosphatonin may be a peptide with a molecular mass of

8 to 12 kDa that does not alter glucose or alanine transport,

but inhibits sodium-dependent phosphate transport in a

cyclic AMP-independent fashion. However, studies, which

document the presence in various disease states of additional

phosphate transport inhibitors (and stimulants), indicate that

the tumor-induced osteomalacia syndrome may be hetero-

geneous. In this regard, excessive tumor production and

secretion of FGF-23 ( White et al. , 2001b ) and matrix extra-

cellular phosphoglycoprotein (MEPE) have been identified

in large numbers of patients with tumor-induced osteoma-

lacia. Moreover, secreted frizzled related protein 4 (sFRP4)

has recently been identified as a putative phosphatonin

in patients with TIO. Indeed, the gene for this protein was

reported as a highly expressed transcript in hemangiopericy-

tomas that caused TIO and a differential expression cloning

approach also identified sFRP4 as highly expressed in TIO

tumors. Thus, it seems certain that ectopic hormone produc-

tion of a phosphatonin by a tumor underlies the syndrome.

As a result, suspicion of the syndrome is often confirmed by

measurement of serum FGF-23. Indeed, Imel et al. (2006)

studied 22 patients with suspected TIO, 13 of whom had

confirmed tumors. They found elevated FGF-23 concen-

trations in 16 of the 22 patients, using the Immunotopics

C-terminal assay (sensitivity 73%), in 5 of the 22 patients

using the Immunotopics Intact assay (sensitivity 23%), and

in 19 of the 22 patients using the Kainos Intact assay (sen-

sitivity 86%). In the 13 patients with confirmed tumors, the

sensitivity was higher with all assays. Of course, the pres-

ence of normal FGF-23 levels in some patients with putative

TIO suggests that FGF-23 is not responsible for the hypo-

phosphatemia in all affected patients.

In contrast to these observations, patients with TIO sec-

ondary to hematogenous malignancy manifest abnormalities

of the syndrome due to a distinctly different mechanism.

In these subjects the nephropathy induced with light-chain

proteinuria or other immunoglobulin derivatives results in

the decreased renal tubular reabsorption of phosphate char-

acteristic of the disease. Thus, light-chain nephropathy must

be considered a possible mechanism for the TIO syndrome.

Treatment

The first and foremost treatment of TIO is complete resec-

tion of the tumor. However, recurrence of mesenchymal

tumors, such as giant-cell tumors of bone, or inability to

resect certain malignancies completely, such as prostate

carcinoma, has resulted in the development of alternative

therapeutic intervention for the syndrome. In this regard,

administration of calcitriol alone or in combination with

phosphorus supplementation has served as effective ther-

apy for TIO. Doses of calcitriol required range from 1.5 to

3.0 µ g/day, whereas those of phosphorus are 2 to 4 g/day.

Although little information is available regarding the long-

term consequences of such treatment, the high doses of

medicine required raise the possibility that nephrolithia-

sis, nephrocalcinosis, and hypercalcemia may frequently


complicate the therapeutic course. Indeed, hypercalcemia

secondary to parathyroid hyperfunction has been docu-

mented in at least five treated subjects. All of these patients

received phosphorus as part of a combination regimen,

which may have stimulated parathyroid hormone secretion

and led to parathyroid autonomy. Thus, a careful assess-

ment of parathyroid function, serum and urinary calcium,

and renal function is essential to ensure safe and effica-

cious therapy.

In an effort to diminish the complications of high-dose

calcitriol and phosphorus therapy, addition of cinacalcet

to the therapeutic regimen has been tested ( Geller et al. ,

2007 ). The resultant decrease of serum PTH resulted in an

increase of renal phosphate reabsorption and serum phos-

phorus and allowed for a decrease in phosphate supple-

mentation to a dose that was more tolerable. Moreover,

one such treated patient exhibited significant bone healing,

indicated by resolution of activity on a bone scan and lack

of osteomalacia as assessed by histomorphometry.

Dent’s Disease (X-Linked Recessive Hypophosphatemic Rickets)

In the past several decades, multiple syndromes have been

described that are characterized by various combinations of

renal proximal tubular dysfunction (including renal phos-

phate wasting), proteinuria, hypercalciuria, nephrocalcino-

sis, nephrolithiasis, renal failure, and rickets; these disorders,

referred to as Dent’s disease, include X-linked recessive

hypophosphatemic rickets (see Table III ), X-linked recessive

nephrolithiasis with renal failure, and low-molecular-weight

proteinuria with nephrocalcinosis. The spectrum of pheno-

typic features in these diseases is remarkably similar, except

for differences in the severity of bone deformities and renal

impairment. The observation that all of these syndromes are

caused by mutations affecting a chloride channel clarifies

the interrelationship between them and establishes that they

are variants of a single disease ( Scheinman, 1998 ).

Urinary loss of low-molecular-weight proteins is

the most consistent abnormality in the disease, present

in all affected males and in almost all female carriers of

the disorder ( Wrong et al. , 1994 ; Reinhart et al. , 1995 ).

Other signs of impaired solute reabsorption in the proxi-

mal tubule, such as renal glycosuria, aminoaciduria, and

phosphate wasting, are variable and often intermittent.

Hypercalciuria is an early and common feature, whereas

hypokalemia occurs in some patients. Urinary acidification

is normal in over 80% of affected subjects, and when it is

abnormal, the defect has been attributed to hypercalciuria

or nephrocalcinosis ( Wrong et al. , 1994 ; Reinhart et al. ,

1995 ; Buckalew et al. , 1974 ). The disease apparently does

not recur after kidney transplantation ( Scheinman, 1998 ).

These phenotypic features indicate the presence of prox-

imal tubular dysfunction but do not suggest its pathophysi-

ologic basis. The gene causing Dent’s disease, CLCN5 ,

encodes the chloride/proton antiporter, CLC-5, which is

expressed predominantly in the kidney and in particular the

proximal tubule, the thick ascending limb of Henle, and the

alpha intercalated cells of the collecting duct ( Scheinman,

1998 ; Lloyd et al. , 1996 ; Hoopes et al. , 1998 ; Igarashi et al. ,

1998 ). Mapping studies have established linkage of this

gene to the short arm of the X chromosome (Xp11.22;

Scheinman et al. , 1997 ). The gene is a member of a fam-

ily of genes encoding voltage-gated chloride channels and

is critical for acidification in the endosomes that participate

in solute reabsorption and membrane recycling in the proxi-

mal tubule. CLC-5 also alters membrane trafficking via the

receptor-mediated-endocytic pathway that involves megalin

and cubulin. CLC-5 mutations associated with Dent’s dis-

ease impair chloride flow and likely lead to impaired acidi-

fication of the endosomal lumen, and thereby disruption

of endosome trafficking to the apical surface, resulting in

abnormal solute reabsorption by the renal tubule.

It is not clear how this process leads to the increased

intestinal calcium absorption and high serum 1,25(OH) 2 D

levels in this disorder ( Reinhart et al. , 1995 ), because the

25(OH)D-1 α -hydroxylase that catalyzes calcitriol forma-

tion is located in the mitochondria of proximal tubular

cells, whereas C1C-5 is expressed in the thick ascending

limb of Henle’s loop ( Devuyst et al. , 1999 ), a major site

of renal calcium reabsorption. The role of this channel in

the reabsorption of calcium in the thick ascending limb

remains unknown.

Fanconi Syndrome

Rickets and osteomalacia are frequently associated with

Fanconi syndrome, a disorder characterized by phosphatu-

ria and consequent hypophosphatemia, aminoaciduria, renal

glycosuria, albuminuria, and proximal renal tubular aci-

dosis ( De Toni, 1933 ; McCune et al. , 1943 ; Brewer, 1985 ;

Chan and Alon, 1985 ; Chesney, 1990 ). Damage to the renal

proximal tubule, secondary to genetic disease (see Table II )

or environmental toxins, represents the common underlying

mechanism of this disease. Resultant dysfunction results

in renal wasting of those substances primarily reabsorbed

at the proximal tubule. The associated bone disease in this

disorder is likely secondary to hypophosphatemia and/or

acidosis, abnormalities that occur in association with aber-

rantly (Fanconi syndrome, type I) or normally regulated

(Fanconi syndrome, type II) vitamin D metabolism.

Type I

The type I disease resembles in many respects the more

common genetic disease, X-linked hypophosphatemic

rickets (see Table III ). In this regard, the occurrence of

abnormal bone mineralization appears dependent on the

prevailing renal phosphate wasting and resultant hypophos-

phatemia. Indeed, subtypes of the disease 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) 2 D levels that are overtly decreased or abnor-

mally low relative to the prevailing serum phosphorus con-

centration ( Chesney et al. , 1984 ). Although the aberrantly

regulated calcitriol biosynthesis may be due to the abnor-

mal renal phosphate transport, proximal tubule damage and

acidosis may play important roles.

A notable difference between this syndrome and XLH is

a common prevailing acidosis, which may contribute to the

bone disease. In this regard, several studies indicate that aci-

dosis may exert multiple deleterious effects on bone. Such

negative sequelae may be related to the loss of bone cal-

cium that occurs due to calcium release for use in buffering.

Alternatively, several investigators have reported that acido-

sis may impair bone mineralization secondary to the direct

inhibition of renal 25(OH)D-1 α -hydroxylase activity. Others

dispute these findings and claim that acidosis does not cause

rickets or osteomalacia in the absence of hypophosphatemia.

Most likely, hypophosphatemia and abnormally regulated

vitamin D metabolism are the primary factors underlying

rickets and osteomalacia in this form of the disease.

Type II

Tieder et al. (1988) have described two siblings (from a con-

sanguineous mating) who presented with classic characteris-

tics 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 concen-

tration) serum 1,25(OH) 2 D levels and consequent hypercal-

ciuria (see Table III ). Moreover, treatment with phosphate

reduced the serum calcitriol in these patients into the nor-

mal range and normalized the urinary calcium excretion.

In many regards, this syndrome resembles HHRH and rep-

resents 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. Further

studies will be necessary to confirm this possibility.

Treatment

Ideal treatment of the bone disease in this disorder is cor-

rection of the pathophysiological defect influencing proxi-

mal 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 pharmacologi-

cal means (such as in cystinosis and Wilson’s syndrome)

have met with variable success. Indeed, no evidence

exists that indicates whether the proximal tubule dam-

age is reversible on relief of an acute toxicity. Thus, for

the most part, therapy of this disorder must be directed

at raising the serum phosphorus concentration, replacing

calcitriol (in type I disease) and reversing an associated

acidosis. However, use of phosphorus and calcitriol in

this disease has been limited. In general, such replace-

ment therapy leads to substantial improvement or resolu-

tion of the bone disease ( Schneider and Schulman, 1983 ).

Unfortunately, growth and developmental abnormalities,

more likely associated with the underlying genetic disease,

remain substantially impaired. More efficacious therapy,

therefore, is dependent on future research into the causes

of the multiple disorders that cause this syndrome.

Tumoral Calcinosis

Tumoral calcinosis, also referred to as hyperphosphatemic

familial tumoral calcinosis, is a rare genetic disease char-

acterized by periarticular cystic and solid tumorous cal-

cifications. Biochemical markers of the disorder include

hyperphosphatemia and a normal or an elevated serum

1,25(OH) 2 D concentration (see Table III ). Using these cri-

teria, evidence has been presented for autosomal recessive

inheritance of this syndrome. However, an abnormality of

dentition, marked by short bulbous roots, pulp stones, and

radicular dentin deposited in swirls, is a phenotypic marker

of the disease that is variably expressed ( Lyles et al. , 1985 ).

Thus, this disorder may have multiple formes frustes that

could complicate genetic analysis. Indeed, using the dental

lesion, as well as the more classic biochemical and clinical

hallmarks of the disease, an autosomal dominant pattern of

transmission has been documented.

The hyperphosphatemia characteristic of the disease

results from an increase in capacity of renal tubular phos-

phate reabsorption. Hypocalcemia is not a consequence of

this abnormality, however, and the serum parathyroid hor-

mone concentration is normal. Moreover, the phosphaturic

and urinary cAMP responses to parathyroid hormone are not

disturbed. Thus, the defect does not represent renal insen-

sitivity to parathyroid hormone, or hypoparathyroidism.

Rather, the genetic basis for the disease has recently been

revealed with inactivating mutations encoding the FGF-23 ,

UDP- N -galactosamine polypeptide N -acetylgalactosaminyl

transferase 3 ( GALNT3 ), or Klotho genes.

l The mutations of FGF-23 lead to destabilized protein

and resultant increased intracellular proteolysis of

FGF-23, most likely due to furin-like proteases ( Araya

et al., 2005 ). This process increases circulating levels

of the bioinactive C-terminal fragment of FGF-23 and

markedly decreases concentration of the bioactive

intact molecule. Such absence of FGF-23 bioactivity

results in the enhanced renal phosphate reabsorption

and hyperphosphatemia observed in affected patients.

Indeed, several investigators have discovered that FGF-

23 gene knockout mice develop hyperphosphatemia

and severe soft-tissue calcification as expected.

l GALNT3 is a Golgi-associated enzyme that initiates

O -glycosylation of mature polypeptides This enzyme


selectively O -glycosylates a furin-like convertase

recognition sequence in FGF-23, thereby impairing

the normal function of this protein ( Ichikawa et al.,

2005 ; Specktor et al., 2006 ). This process decreases

circulating intact, bioactive FGF-23 as described earlier

for the destabilizing mutations of FGF-23.

l Recent studies revealed that FGF-23 requires an

additional cofactor, klotho (KL), to bind and signal

through its cognate FGF receptors. Not surprisingly,

therefore, diminished KL expression in mice results in a

phenotype characterized by severe hyperphosphatemia,

increased serum 1,25(OH) 2 D levels, and ectopic

vascular and soft-tissue calcifications. Indeed the

KL -deficient phenotype broadly overlaps with the

phenotype of Fgf-23 -null mice, reaffirming the

functional crosstalk between KL and FGF-23 and

underscoring the observed interactions between KL,

FGF-23 and its cognate FGF receptors. In accord,

Ichikawa et al. (2007) have recently reported a point

mutation in the KL gene, which attenuates the ability

of KL to support FGF-23 signaling and thereby causes

the deranged phosphate, vitamin D and calcium

homeostasis, characteristic of tumoral calcinosis.

Clearly, therefore, the basis of the disease is diminished

FGF-23 function, which results in enhanced renal phos-

phate reabsorption and increased calcitriol production.

Undoubtedly, calcific tumors result from the elevated cal-

cium–phosphorus product. The observation that long-term

phosphorus depletion alone or in association with admin-

istration of acetazolamide, a phosphaturic agent, leads to

resolution of the tumor masses supports the assumption

that an increased calcium–phosphorus product underlies

the calcific tumor formation.

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, cal-

citriol, or high-calcium-content dialysates. Calcific tumors

again likely result from an elevated calcium–phosphorus

product. Indeed, complete remission of the tumors occurs

on treatment with vinpocetine, a mineral scavenger drug,

dialysis with low-calcium-content dialysate, and renal

transplantation

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Chapter 1

INTRODUCTION

Magnesium (Mg) is the fourth most abundant cation and

the second most abundant intracellular cation in vertebrates.

Mg is involved in numerous biological processes and is

essential for life ( Rude and Shils, 2006 ). This mineral has

evolved to become a required cofactor in literally hun-

dreds of enzyme systems ( Laires et al., 2004 ; Maguire

and Cowan, 2002 ; Romani, 2006 ; Rude and Shils, 2006 ).

Examples of the physiological role of Mg are shown in

( Table I ). Mg may be required for enzyme substrate forma-

tion. For example, enzymes that utilize ATP do so as the

metal chelate, MgATP. Free Mg 2 1 also acts as an allosteric

activator of numerous enzyme systems, as well as playing

a role in ion channels and for membrane stabilization. Mg

is therefore critical for a great number of cellular functions,

Chapter 24

including oxidative phosphorylation, glycolysis, DNA tran-

scription, and protein synthesis.

MAGNESIUM METABOLISM

The normal adult total body Mg content is approximately

25 grams (2000 mEq or 1 mol) of which 50% to 60%

resides in bone ( Elin, 1987 ; Wallach, 1988 ). Mg constitutes

0.5% to 1% of bone ash (200 mmol/kg ash weight). One-

third of skeletal Mg is surface limited and exchangeable,

and this fraction may serve as a reservoir for maintaining

a normal extracellular Mg concentration ( Wallach, 1988 ;

Rude and Shils, 2006 ; Laires et al., 2004 ). The remainder

of Mg in bone is an integral component of the hydroxyapa-

tite lattice, which may be released during bone resorption.

The rest of body Mg is mainly intracellular. The Mg content

of soft tissues varies between 6 and 25 mEq/kg wet weight

( Elin, 1987 ; Rude and Shils, 2006 ). In general, the higher

the metabolic activity of the cell, the higher the Mg con-

tent. The concentration of Mg within cells is in the order of

5–20 mmol/liter, of which 1% to 5% is ionized or free ( Elin,

1987 ; Romani, 2006 ; Rude and Shils, 2006 ). The distribu-

tion of Mg in the body is shown in ( Table II ) and ( Fig. 1 ).

Extracellular Mg accounts for about 1% of total body

Mg. Mg concentration or content may be reported as mEq/

liter, mg/dl, or mmol/liter. Values reported as mEq/liter can

be converted to mg/dl by multiplying by 1.2 and to mmol/

liter by dividing by 0.5 . The normal serum Mg concentration

is 1.5–1.9 mEq/liter (0.7–1.0 mmol/liter) ( Elin, 1987 ; Rude

and Shils, 2006 ). About 70% to 75% of plasma Mg is ultra-

filterable, of which the major portion (55% of total serum

Mg) is ionized or free, and the remainder is complexed to

citrate, phosphate, and other anions as represented schemat-

ically in ( Fig. 2 ). The remainder is protein bound; 25% of

total serum Mg is bound to albumin and 8% to globulins.

Intestinal Mg Absorption

Mg homeostasis is dependent on the amount of Mg ingested,

efficiency of Mg intestinal absorption, and renal reabsorption

and excretion. Intestinal Mg absorption is proportional

Magnesium Homeostasis

Robert K. Rude, M.D. Keck School of Medicine, University of Southern California Los Angeles, California

TABLE I Examples of the Physiological Role of

Magnesium

I. Enzyme substrate (ATP Mg, GTP Mg)

A. ATPase or GTPase (Na 1 , K 1 -ATPase, Ca 2 1 -ATPase)

B. Cyclases (adenylate cyclase, guanylate cyclase)

C. Kinase (hexokinase, creatine kinase, protein kinase)

II. Direct enzyme activation

A. Adenylate cyclase

B. Phospholipase C

C. Na 1 , K 1 -ATPase

D. Ca 2 1 -ATPase

E. K 1 , H 1 -ATPase

F. G proteins

G. 5 9 -Nucleotidase

H. Creatine kinase

I. Phosphofructokinase

J. 5-Phosphoribosyl-pyrophosphate synthetase

K. Lipoprotein lipase

III. Infl uence membrane properties

A. K 1 channels

B. Ca 2 1 channels

C. Nerve conduction


TABLE II Distribution of Magnesium in Adult Humans a

Tissue Body mass,

kg (wet wt)

Mg concentration,

mmol/kg (wet wt)

Mg content

(mmol)

% of total

body Mg

Serum 3.0 0.85 2.6 0.3

Erythrocyte 2.0 2.5 5.0 0.5

Soft tissue 22.7 8.5 193.0 19.3

Muscle 30.0 9.0 270.0 27.0

Bone 12.3 43.2 530.1 52.9

Total 70.0 1000.7 100.0

a Adapted from Elin (1987) .

FIGURE 1 Distribution of magnesium in the body.

Soft tissues Skeleton

54

% (

1,0

80

mE

q)

45

% (

90

0 m

Eq

)

1% (20 mEq) Extracellular fluid

Proteinbound

Free

Complexed

15% (0.1 6 .02 mEq/L)

30

% (

0.6

6 0

.1 m

Eq

/L)

55

% (

1.1

6 0

.1 m

Eq

/L)

FIGURE 2 Physicochemical states of magnesium in normal plasma.

et al. , 2002 ; Schlingmann et al. , 2004 ). This gene encodes

for TMP6 in the intestinal epithelium for an Mg perme-

able channel ( Schlingmann and Gudermann, 2005 ), which

is evidence for at least one active Mg transport process.

TMPR7 has also been demonstrated to play a role in Mg

transport ( Schlingmann and Gudermann, 2005 ; Chubanov

et al., 2004 ). Under normal dietary conditions in healthy

individuals, approximately 30% to 50% of ingested Mg

is absorbed ( Brannan et al., 1976 ; Hardwick et al., 1990 ;

Fine et al., 1991 ; Kayne and Lee, 1993 ; Schweigel and

Martens, 2000 ). Mg is absorbed along the entire intestinal

tract, including the large and small bowel, but the sites of

maximal Mg absorption appear to be the ilium and distal

jejunum ( Hardwick et al., 1990 ; Fine et al., 1991 ; Kayne

and Lee, 1993 ; Schweigel and Martens, 2000 ).

The recommended daily allowance for Mg is 420 mg per

day for adult males and 320 mg per day for adult females

to the amount ingested ( Fine et al., 1991 ; Schweigel and

Martens, 2000 ). The mechanism(s) for intestinal Mg

absorption includes passive diffusion and active transport

( Fine et al., 1991 ; Kayne and Lee, 1993 ; Bijvelds et al.,

1998 ; Schweigel and Martens, 2000 ). Shown in ( Fig. 3 )

is a schematic model ( Schlingmann et al. , 2004 ) as sug-

gested by rat ( Ross, 1962 ) and human studies ( Fine et al.,

1991 ; Kayne and Lee, 1993 ), which may account for the

higher fractional intestinal Mg absorption at low dietary

Mg intakes. Others have concluded that intestinal Mg

absorption in humans increases linearly with Mg intake (for

review, see Schweigel and Martens, 2000 ). The report of a

patient with primary hypomagnesemia who was shown to

malabsorb Mg during low Mg concentration in the intestine

suggested an active transport process ( Milla et al., 1979 ). A

familial defect in intestinal Mg absorption has been mapped

to chromosome 9q22 ( Walder et al., 1997 ; Schlingmann

489Chapter | 24 Magnesium Homeostasis

( Institute of Medicine, 1997 ). The dietary Mg intake in

Western culture, however, appears to fall below that in

a large section of the population across all ages, ranging

from approximately 150 to 350 mg per day, suggesting that

occult Mg depletion may be relatively prevalent ( Morgan

et al., 1985 ; Marier, 1986 ). A recent study, however, has

suggested that the dietary Mg requirement may be lower

( Hunt and Johnson, 2006 ). The major sources of Mg are

nuts, cereals, green leafy vegetables, and meats.

A principal factor that regulates intestinal Mg transport

has not been described. Vitamin D, as well as its metabo-

lites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D

[1,25(OH) 2 D] have been observed in some studies to

enhance intestinal Mg absorption but to a much lesser extent

than they do calcium absorption ( Brannan et al., 1976 ;

Hodgkinson et al., 1979 ; Krejs et al., 1983 ). Although net

intestinal calcium absorption in humans correlates with

plasma 1,25(OH) 2 D concentrations, Mg does not ( Wilz

et al., 1979 ). A low Mg diet has been shown to increase

intestinal calbindin-D 9k , suggesting that this vitamin

D-dependent, calcium-binding protein may play a role in

intestinal Mg absorption ( Hemmingsen et al., 1994 ).

Bioavailability of Mg may also be a factor in Mg intesti-

nal absorption as other nutrients may affect Mg absorption.

Although dietary calcium has been reported to both decrease

and increase Mg absorption, human studies have shown no

effect ( Brannan et al., 1976 ; Fine et al., 1991 ). The presence

of excessive amounts of substances such as free fatty acids,

phytate, oxalate, polyphosphates, and fiber may bind Mg

and impair absorption ( Seelig, 1981 ; Franz, 1989 ).

Renal Mg Handling

The kidney is the principal organ involved in Mg homeo-

stasis ( Cole and Quamme, 2000 ; Satoh and Romero, 2002 ).

During Mg deprivation in normal subjects, the kidney

conserves Mg avidly and less than 1–2 mEq is excreted in

the urine per day ( Barnes et al., 1958 ). Conversely, when

excess Mg is taken, it is excreted into the urine rapidly

( Heaton and Parson, 1961 ). The renal handling of Mg in

humans is a filtration–reabsorption process; there appears

to be no tubular secretion of Mg. Micropuncture studies of

the nephron in several mammalian species have indicated

that Mg is absorbed in the proximal tubule, thick ascend-

ing limb of Henle, and distal convoluted tubule ( Quamme

and De Rouffignac, 2000 ; Cole and Quamme, 2000 ), as

illustrated in ( Fig. 4 ).

About 2,400 mg of Mg is filtered daily through the

normal adult. Of this, approximately 15% to 20% of fil-

tered Mg is reabsorbed in the proximal convoluted tubule.

Current data suggest that Mg transport in this segment is

reabsorbed passively through the paracellular pathway

( Cole and Quamme, 2000 ; Satoh and Romero, 2002 ). The

majority, approximately 65% to 75%, of filtered Mg is

reclaimed in the loop of Henle with the major site at the

cortical thick ascending limb. Magnesium transport in this

segment appears to be dependent on the transepithelial

potential generated by NaCl absorption ( Cole and Quamme,

2000 ; Satoh and Romero, 2002 ). Mutation of the CLDN16

gene, which encodes for a defect in paracellin-1 in the thick

asending limb of Henle, results in renal Mg and calcium

wasting ( Schlingmann et al., 2002 ; Konrad et al., 2004 ).

Micropuncture studies have also demonstrated that hyper-

magnesemia or hypercalcemia will decrease Mg reabsorp-

tion in this segment independent of NaCl transport ( Cole

and Quamme, 2000 ). Studies suggest that the concentration

of calcium and/or Mg in the extracellular fluid may regulate

absorption of Mg in the thick ascending limb of Henle by

activation of the Ca 2 1 -sensing receptor in this segment of

the nephron ( Brown and Hebert, 1996 ; Cole and Quamme,

2000 ). Inactivating and activating mutations of the Ca 2 1 -

sensing receptor will result in hypermagnesemia and hyper-

calcemia with decreased urine excretion of both cations or

hypomagnesemia and hypocalcemia with increased excre-

tion of both cations, respectively ( Konrad et al., 2004 ).

Approximately 5% to 10% of Mg is reclaimed in the dis-

tal tubule where reabsorption is trancellular and active in

TRPM6

Lumen

?

Blood

Na1

3 Na1

Mg21

Mg21

Mg21

Mg21

HSH

2K1ATPase

A

Mg intake

Transcellular

Paracellular

CombinedPhysiologic

range

Ne

t M

g a

bso

rptio

n

B

FIGURE 3 A . Schematic model of intestinal magnesium absorption

via two independent pathways: passive absorption via the paracellular

pathway and active transcellular transport consisting of an apical entry

through a putative magnesium channel and a basolateral exit mediated

by a putative sodium-coupled exchange. B. Kinetics of human intestinal

magnesium absorption. Paracellular transport linearly rising with intralu-

minal concentration (dotted line) and saturable active trancellular trans-

port (dashed line) together yield a curvilinear function for net magnesium

absorption (solid line). ( Schlingmann et al., 2004 , with permission).


nature ( Cole and Quamme, 2000 ). A genetic defect on chro-

mosome 11q23, gene FXYD2 results in a defect in Na-K-

ATPase resulting in renal Mg wasting ( Konrad et al. , 2004 ;

Meij et al ., 2002 ). TRPM6, TRMP7, and TRMP9, members

of the TRMP family of cation channels, may also affect Mg

transport as genetic defects of these proteins have resulted

in renal Mg wasting ( Schmitz et al ., 2003 ; Schlingmann

et al., 2002 ; Schlingmann et al., 2004 ). It is speculated that

transport at this site may be regulated hormonally and serve

to finely regulate Mg homeostasis.

Micropuncture studies performed during a time in

which the concentration of Mg was increased gradually, in

either the tubular lumen or in the extracellular fluid, have

failed to demonstrate a tubular maximum for Mg (TmMg)

in the proximal tubule ( Quamme and De Rouffignac, 2000 ).

The rate of Mg reabsorption is dependent on the concentra-

tion of Mg in the tubule lumen. Similarly, a TmMg was not

reached in the loop of Henle during a graduated increase

in the luminal-filtered Mg load. Hypermagnesemia, as dis-

cussed earlier, however, results in a marked depression of

Mg resorption in this segment. In vivo studies in animals

and humans, however, have demonstrated a TmMg that

probably reflects a composite of tubular reabsorption pro-

cesses, as shown in Fig. 5 ( Rude et al., 1980 ; Rude and

Ryzen, 1986 ).

During Mg deprivation, Mg virtually disappears from

the urine ( Barnes et al., 1958 ). Despite the close regulation

of Mg by the kidney, there has been no hormone or fac-

tor described that is responsible for renal Mg homeostasis.

Micropuncture studies have shown that PTH changes the

potential difference in the cortical thick ascending limb and

increases Mg reabsorption ( Quamme and De Rouffignac,

2000 ; Cole and Quamme, 2000 ). When given in large

doses in humans or other species, PTH decreases urinary

Mg excretion ( Bethune et al., 1968 ; Massry et al., 1969 ).

However, patients with either primary hyperparathyroidism

or hypoparathyroidism usually have a normal serum Mg

concentration and a normal TmMg, suggesting that PTH

is not an important physiological regulator of Mg homeo-

stasis ( Rude et al., 1980 ). Glucagon, calcitonin, and ADH

also affect Mg transport in the loop of Henle in a manner

similar to PTH ( Quamme and De Rouffignac, 2000 ; Cole

and Quamme, 2000 ); the physiological relevance of these

actions is unknown. Little is known about the effect of

vitamin D on renal Mg handling. An overall view of Mg

metabolism is shown in ( Fig. 6 ) .

Intracellular Mg

Within the cell, Mg is compartmentalized and most of it is

bound to proteins and negatively charged molecules such

as ATP, ADP, RNA, and DNA; in the cytoplasm, about

80% of Mg is complexed with ATP ( Gupta and Moore,

1980 ; Frausto da Silva and Williams, 1991; Romani, 2006 ).

Significant amounts of Mg are found in the nucleus, mito-

chondria, and endoplasmic and sarcoplasmic reticulum as

well as in the cytoplasm ( Gunther, 1986 ; Romani, 2006 ).

Proximal convoluted tubule Loop of Henle Distal convoluted tubule

5–10%

65–75%

15–20%

3–5%

Collectingduct

Thickascendinglimb

Thindescending

limb

Medullary

100%Mg

Cortical

FIGURE 4 Summary of the tubular handling of magnesium. Schematic illustration of the cellular transport of magnesium within the thick ascending

limb of the loop of Henle ( Cole and Quamme, 2000 ).


Total cell Mg concentration has been reported to range

between 5 and 20 m M ( Gunther, 1986 ; Romani, 2006 ).

The concentration of free ionized Mg 2 1 , which has been

measured in the cytoplasm of mammalian cells, has ranged

from 0.2 to 1.0 m M, depending on cell type and means of

measurement ( Raju et al., 1989 ; London, 1991 ; Romani

and Scarpa, 1992 ; Romani, 2006 ). It constitutes 1% to 5%

of the total cellular Mg. The Mg 2 1 concentration in the

cell cytoplasm is maintained relatively constant even when

the Mg 2 1 concentration in the extracellular fluid is experi-

mentally varied to either high or low nonphysiological lev-

els ( Dai and Quamme, 1991 ; Quamme et al., 1993 ; Romani

et al., 1993 ). The relative constancy of the Mg 2 1 in the intra-

cellular milieu is attributed to the limited permeability of the

plasma membrane to Mg and to the operation of specific

Mg transport systems, which regulate the rates at which Mg

is taken up by cells or extruded from cells ( Flatman, 1984 ;

Romani, 2006 ; Murphy, 2000 ). Although the concentration

differential between the cytoplasm and the extracellular fluid

for Mg 2 1 is minimal, Mg 2 1 enters cells down an electro-

chemical gradient because of the relative electronegativity of

the cell interior. Maintenance of the normal intracellular con-

centrations of Mg 2 1 requires that Mg be actively transported

out of the cell ( Murphy, 2000 ; Romani, 2006 ).

Studies in mammalian tissues and isolated cells sug-

gest the presence of specific Mg transport systems. Early

in vivo studies, using the radioactive isotope 28 Mg, sug-

gested that tissues vary with respect to the rates at which

Mg exchange occurs and the percentage of total Mg that is

readily exchangeable ( Rogers and Mahan, 1959 ). The rate

of Mg exchange in heart, liver, and kidney exceeded that in

skeletal muscle, red blood cells, brain, and testis ( Romani,

2006 ). These studies do show that, albeit slow in some tis-

sues, there is a continuous equilibration of Mg between

cells and the extracellular fluid. Buffering of intracellular

Mg by ATP and other negatively charged molecules may

also play a role in maintaining a constant intracellular Mg

concentration ( Romani, 2006 ). An increased cellular Mg

content has been reported for rapidly proliferating cells,

indicating a possible relationship between the metabolic

state of a cell and the relative rates of Mg transport into

and out of cells ( Cameron et al., 1980 ).

Mg transport out of cells appears to require the presence

of carrier-mediated transport systems, possibly regulated by

the concentration of Mg 2 1 within the cell ( Romani, 2006 ;

Gunther, 1993 ; Gunther, 2006 ). The efflux of Mg from the

cell is coupled to Na transport and requires energy ( Romani,

2006 ; Gunther, 1993 ; Murphy, 2000 ; Gunther, 2006 ).

Muscle tissue, which is incubated in isotonic sucrose, a low

sodium buffer, or in the presence of ouabain as a metabolic

inhibitor, has been shown to accumulate large amounts of

Mg. These studies also suggest that the efflux of Mg from

the cell is coupled with the movement of sodium down its

electrochemical gradient into the cell. Maintenance of this

process would require the subsequent extrusion of sodium

by the Na 1 , K 1 -ATPase. There is also evidence for a Na-

independent efflux of Mg, however ( Gunther, 1993, 2006 ).

Mg influx appears to be linked to Na and HCO 3 transport,

but by a different mechanism than efflux ( Gunther, 1993 ;

Gunther and Hollriegl, 1993 ; Romani, 2006 ). The molecu-

lar characteristics of the Mg transport proteins have not

been described. Studies in prokaryotes, however, have iden-

tified three separate transport proteins for Mg ( Smith and

Maguire, 1993 ). TRPM6, TRPM7, and TRMP9, members

of the TRPM family of cation channels, have also been

demonstrated to influence Mg transport across the plasma

membrane ( Schmitz et al ., 2003 ; Schlingmann et al ., 2002 ,

Schlingmann et al. , 2004).

Mg transport in mammalian cells is influenced by hor-

monal and pharmacological factors. Mg 2 1 efflux from

isolated perfused rat heart and liver ( Romani and Scarpa,

1990 ; Gunther et al., 1991 ; Gunther, 1993 ) or thymocytes

Normal

Bisector

Slope 1.00

TmMg

4.0

3.0

2.0

1.0

1.0 2.0 3.0 4.0 5.0

Ultrafiltrable serum magenesium mg/100ml

Ma

gn

esiu

m e

xcre

tio

n m

g/1

00

ml

G.F

./1

.73

sq

. m

ete

r

FIGURE 5 Urinary magnesium excretion is plotted against ultrafiltra-

ble serum Mg in normal subjects before and during magnesium infusion.

Data are related to a bisector that corresponds to the theoretical value

of magnesium excretion if no magnesium were reabsorbed ( Rude et al.,

1980 ).

300mg

100mg

2,300mg

2,400mg

12mg12mg

200mg

100mg

Kidney

Plasma

Bone

Inte

stine

FIGURE 6 Schematic representation of magnesium metabolism.


( Gunther and Vormann, 1990 ) is stimulated after short-

term acute exposure to β -agonists and permeant cAMP.

Because intracellular Mg 2 1 does not change, a redistribu-

tion from the mitochondria was suggested, as cAMP can

induce Mg 2 1 release from this compartment ( Romani and

Scarpa, 1992 ) or by altered buffering of Mg within the cell

( Murphy, 2000 ). In contrast, Mg 2 1 influx was stimulated

by β -agonists after a more prolonged exposure in hepato-

cytes, as well as in adipocytes and vascular smooth mus-

cle, presumably mediated by protein kinase A ( Zama and

Towns, 1986 ; Ziegler et al., 1992 ; Gunther, 1993 ; Romani

et al., 1993 ). However, the rate of Mg uptake by the mouse

lymphoma S49 cell line is inhibited by β -adrenergic

agents ( Maguire, 1984 ). Activation of protein kinase C by

diacyl-glycerol or by phorbol esters also stimulates Mg 2 1

influx and does not alter efflux ( Grubbs and Maguire,

1986 ; Romani, 2006 ). Mg 2 1 extrusion is also elicited by

~ 1-adrenergic receptors in a cAMP-independent manner

( Fagen and Romani, 2001 ; Romani, 2006 ).

Growth factors may also influence Mg 2 1 uptake

by cells. Epidermal growth factor has been shown to

increase Mg transport into a vascular smooth muscle cell

line ( Grubbs, 1991 ). Insulin and dextrose were found to

increase 28 Mg uptake by a number of tissues, including

skeletal and cardiac muscle, in which total cellular Mg con-

tent increased as well ( Lostroh and Krahl, 1973 ). Increased

amounts of total intracellular Mg following treatment with

insulin in vitro have been reported in uterine smooth muscle

and chicken embryo fibroblasts ( Aikawa, 1960 ; Sanui and

Rubin, 1978 ). An insulin-induced transport of Mg into cells

could be one factor responsible for the fall in the serum Mg

concentration observed during insulin therapy of diabetic

ketoacidosis ( Kumar et al., 1978 ). The effect of insulin on

total cellular Mg may differ from its effects on intracellular-

free Mg 2 1 . Measurements of intracellular-free Mg 2 1 in frog

skeletal muscle failed to show an effect of insulin ( Gupta

and Moore, 1980 ); however, other studies demonstrated that

insulin increases Mg 2 1 in human red blood cells, platelets,

lymphocytes, and heart ( Hwang et al., 1993 ; Barbagallo

et al., 1993 ; Hua et al., 1995 ; Romani et al., 2000 ).

It is hypothesized that this hormonally regulated Mg

uptake system controls intracellular Mg 2 1 concentration in

cellular subcytoplasmic compartments. The Mg 2 1 concen-

tration in these compartments would then serve to regulate

the activity of Mg-sensitive enzymes.

Risk Factors and Causes of Magnesium

Defi ciency

Prevalence

The many risk factors for magnesium depletion shown in

( Table III ) suggest that this condition is not a rare occur-

rence in acutely or chronically ill patients. A survey of

2,300 patients in a Veterans Administration hospital found

6.9% were hypomagnesemic; 11% of patients having rou-

tine magnesium determinations were hypomagnesemic

( Whang et al ., 1984 . Similar high rates of depletion have

been reported in studies of ICU patients ( Whang et al .,

1994 ; Ryzen et al. , 1985 ; Chernow et al., 1989; Fiaccadori

et al., 1988). The true prevalence of magnesium depletion

is not known, because this ion is not included in routine

electrolyte testing in many clinics or hospitals ( Whang

et al ., 1994 ).

Gastrointestinal Disorders

Gastrointestinal disorders ( Table III ) may lead to magne-

sium depletion in various ways. The Mg content of upper

intestinal tract fluids is approximately 1 mEq/L. Vomiting

and nasogastric suction therefore may contribute to Mg

depletion. The Mg content of diarrheal fluids is much

higher (up to 15 mEq/L), and consequently Mg depletion

TABLE III Causes of Magnesium Defi ciency

1. Gastrointestinal Disorders

a. Prolonged nasogastric suction/vomiting

b. Acute and chronic diarrhea

c. Intestinal and biliary fistulas

d. Malabsorption syndromes

e. Extensive bowel resection or bypass

f. Acute hemorrhagic pancreatitis

g. Protein-calorie malnutrition

h. Primary intestinal hypomagnesemia (neonatal)

2. Renal Loss

a. Chronic parenteral fluid therapy

b. Osmotic diuresis (glucose, urea, manitol)

c. Hypercalcemia

d. Alcohol

e. Diuretics (furosemide, ethacrynic acid)

f. Aminoglycosides

g. Cisplatin

h. Cyclosporin

i. Amphotericin B

j. Pentamidine

k. Tacolimus

l. Metabolic acidosis

m. Renal disorders with Mg wasting

n. Primary renal hypomagnesemia

3. Endocrine and Metabolic Disorders

a. Diabetes mellitus (glycosuria)

b. Phosphate depletion

c. Primary hyperparathyroidism (hypercalcemia)

d. Hypoparathyroidism (hypercalciuria,

hypercalcemia owing to overtreatment

with vitamin D)

e. Primary aldosteronism

f. Hungry bone syndrome

g. Chronic renal disease

h. Excessive lactatation


is common in acute and chronic diarrhea, regional enteri-

tis, ulcerative colitis, and intestinal and biliary fistulas

( Thoren, 1963 ; LaSala et al ., 1985 ; Nyhlin et al. , 1985 ).

Malabsorption syndromes towing to nontropical sprue,

radiation injury resulting from therapy for disorders such

as carcinoma of the cervix, and intestinal lymphangiecta-

sia may also result in Mg deficiency ( Booth et al., 1963 ;

Goldman et al., 1962; Habtezion et al., 2002 ). Steatorrhea

and resection or bypass of the small bowel, particularly

the ileum, often results in intestinal Mg loss or malabsorp-

tion ( Dyckner et al., 1982 ; Van Gaal et al., 1987 ). Lastly,

acute severe pancreatitis is associated with hypomagnese-

mia, which may be owing to the clinical problem causing

the pancreatitis, such as alcoholism, or to saponification

of Mg in necrotic parapancreatic fat ( Weir et al. , 1975 ;

Ryzen and Rude, 1990 ). A primary defect in intestinal Mg

absorption, which presents early in life with hypomagnese-

mia, hypocalcemia, and seizures, has been described as an

autosomal recessive disorder linked to chromosome 9q22

as discussed earlier ( Walder et al., 1997 ; Schlingmann

et al ., 2002 ; Schlingmann et al ., 2004 ), a disorder appears

to be caused by mutations in TRPM6, which expresses a

protein involved with active intestinal Mg transport.

Renal Disorders

Excessive excretion of Mg into the urine may be the basis

of Mg depletion ( Table III ). Renal Mg reabsorption is pro-

portional to tubular fluid flow as well as to sodium and

calcium excretion. Therefore, chronic parenteral fluid ther-

apy, particularly with saline, and volume expansion states

such as primary aldosteronism, and hypercalciuric states

may result in Mg depletion ( Massry et al. , 1967 ; Coburn,

1970 ; McNair et al ., 1982 ). Hypercalcemia have been

shown to decrease renal Mg reabsorption probably medi-

ated by calcium binding to the calcium-sensing receptor

in the thick ascending limb of Henle and decreasing tran-

sepithelial voltage and is probably the cause of renal Mg

wasting and hypomagnesemia observed in many hypercal-

cemic states ( Brown and Hebert, 1995 ; Cole and Quamme,

2000 ). Osmotic diuresis because of glucosuria will result

in urinary Mg wasting ( McNair et al ., 1982 ).

Many pharmaceutical drugs may cause renal Mg wast-

ing and Mg depletion. The major site of renal Mg reab-

sorption is at the loop of Henle, therefore diuretics such as

furosemide and ethacrynic acid have been shown to result

in marked Mg wasting ( Quamme, 1981 ). Aminoglycosides

have been shown to cause a reversible renal lesion that

results in hypermagnesuria and hypomagnesemia ( Zaloga

et al ., 1984 ; Kes and Reiner, 1990 ). Similarly, ampho-

tericin B therapy has been reported to result in renal Mg

wasting. Other renal Mg-wasting agents include cisplatin,

cyclosporin, tacrolimus, and pentamidine ( Meyer and

Madias, 1994 ). A rising blood alcohol level has been

associated with hypermagnesuria and is one factor contribut-

ing to Mg depletion in chronic alcoholism. Metabolic acido-

sis because of diabetic ketoacidosis, starvation, or alcoholism

may also result in renal Mg wasting ( Lau et al ., 1987 ).

Several renal Mg wasting disorders have been

described that may be genetic or sporadic as discussed

earlier in renal Mg metabolism (Konrad et al. , 2004; Meij

et al ., 2002 ). Gitelman’s syndrome (familial hypokalemia-

hypomagnesemia syndrome) is an autosomal recessive

disorder owing to a genetic defect of the thiazide-sensi-

tive NaCl cotransporter gene on chromosome 16 ( Simon

et al ., 1996 ). Recently, mutations in the tight-junction

gene Claudin 19 have been associated with renal Mg wast-

ing and renal failure and ocular abnormalities ( Konrad

et al. , 2006 ). Mutations of other cation channels, TMPR6,

TMPR7, and TMPR9, have also recently been associated

with renal Mg wasting ( Schmitz et al ., 2003 ; Schlingmann

et al., 2002 ; Schlingmann et al. , 2004 ). Other undefined

genetic defects also exist ( Kantorovich et al ., 2002 ).

Hypomagnesemia may accompany a number of other

disorders (for review see Rude, 1996 ). Phosphate deple-

tion has been shown experimentally to result in urinary Mg

wasting and hypomagnesemia. Hypomagnesemia may also

accompany the “ hungry bone ” syndrome, a phase of rapid

bone mineral accretion in subjects with hyperparathy-

roidism or hyperthyroidism following surgical treatment.

Finally, chronic renal tubular, glomerular, or interstitial

diseases may be associated with renal Mg wasting. Rarely,

excessive lactation may result in hypomagnesemia.

Diabetes Mellitus

Special consideration must be given to diabetes mellitus.

It is the most common disorder associated with magne-

sium deficiency ( McNair et al ., 1982 ; Rude, 1996 ; Song

et al., 2004 ; Lopez-Ridaura et al ., 2004 ). It is generally

thought that the mechanism for magnesium depletion in

diabetics is owing to renal magnesium wasting secondary

to osmotic diuresis generated by hyperglycosuria. Dietary

magnesium intake, however, falls below the RDA in dia-

betics. Magnesium deficiency has been reported to result

in impaired insulin secretion as well as insulin resistance

( Song et al., 2004 ; Lopez-Ridaura et al ., 2004 ). The mech-

anism is unclear but may be because of abnormal glucose

metabolism as magnesium is a cofactor in several enzymes

in this cycle. In addition, magnesium depletion may

decrease tyrosine kinase activity at the insulin receptor and

magnesium may influence insulin secretion by the beta

cell. Diabetics given magnesium therapy appear to have

improved diabetes control. Recently, two studies have

reported that the incidence of type-2 diabetes is signifi-

cantly greater in people on a lower magnesium diet ( Song

et al., 2004 ; Lopez-Ridaura et al ., 2004 ). Magnesium sta-

tus should therefore be assessed in patients with diabetes


mellitus as a vicious cycle may occur; diabetes out of con-

trol leading to magnesium loss and the subsequent magne-

sium deficiency resulting in impaired insulin secretion and

action and worsening diabetes control.

ROLE OF MAGNESIUM IN BONE AND

MINERAL HOMEOSTASIS

Because of the prevalence of Mg in both cells and bone, as

well as its critical need for numerous biological processes

in the body, it is not surprising that this mineral plays a

profound role in bone and mineral homeostasis. Our under-

standing of the role of magnesium has developed princi-

pally through observations of the effect of Mg depletion

in both humans and animals. Mg influences the formation

and/or secretion of hormones that regulate skeletal homeo-

stasis and the effect of these hormones on bone. Mg can

also directly affect bone cell function as well as influence

hydroxyapatite crystal formation and growth. These areas

are discussed later and are outlined in Table IV .

Parathyroid Hormone Secretion

Calcium is the major regulator of PTH secretion. Mg,

however, modulates PTH secretion in a manner similar

to calcium. A number of in vitro and in vivo studies have

demonstrated that acute elevations of Mg inhibit PTH secre-

tion, whereas an acute reduction stimulates PTH secretion

( Sherwood et al. , 1970 ; Cholst et al., 1984 ; Ferment et al.,

1987 ; Toffaletti et al., 1991 , Rude, 1994 ; Rude and Shils,

2006 ). These data suggest that Mg could be a physiologic

regulator of PTH secretion. Although early investigations

indicated that Mg was equipotent to calcium in its effect on

parathyroid gland function ( Sherwood et al., 1970 ), more

recent studies demonstrated that Mg has approximately

30 to 50% the effect of calcium on either stimulating or

inhibiting PTH secretion ( Wallace and Scarpa, 1982 ; Ferment

et al., 1987 ; Toffaletti et al., 1991 ; Rude, 1994 ). The finding

in humans that a 5% (0.03 m M ) decrease in serum ultrafil-

terable Mg did not result in any detectable change in intact

serum PTH concentration while a 5.5% (.07 m M ) decrease

in ionized calcium resulted in a 400% increase in serum

PTH supports this concept ( Toffaletti et al., 1991 ).

The inhibitory effects of Mg on PTH secretion may

be dependent on the extracellular calcium concentration

( Brown et al., 1984 ). At physiological calcium and Mg

concentrations, these divalent cations were found to be

relatively equipotent at inhibiting PTH secretion from dis-

persed bovine parathyroid cells ( Brown et al., 1984 ). At a

low calcium concentration (0.5 m M ), however, a threefold

greater Mg concentration was required for similar PTH

inhibition. Altering the Mg concentration did not diminish

the ability of calcium to inhibit PTH secretion. Differences

TABLE IV Effect of Mg Depletion on Bone and Mineral Metabolism

Effect Potential mechanism(s)

1.

Decreased PTH

secretion

Altered phosphoinositol activity

Decreased adenylate cyclase activity

2.

Decreased PTH

action

Decreased adenylate cyclase activity

Altered phosphoinositol activity

3.

Decreased serum

1,25(OH) 2 D

Decreased serum PTH

Renal PTH resistance (decrease in

1 α -hydroxylase activity)

4.

Impaired vitamin D

metabolism and action

Decreased 1,25(OH) 2 D formation

Decreased intestinal epithelial cell and

osteoblast activity

Skeletal resistance to vitamin D

5.

Impaired bone

growth/osteoporosis

Decreased PTH and 1,25(OH) 2 D formation

and action

Decreased effect of insulin and IGF-1

Direct effect to decrease bone cell activity

6.

Increased bone

resorption/osteoporosis

Increased cytokine production

Increased substance P, TNF α , IL-1, IL-6

Increased RANKL, decreased OPG

7.

Altered hydroxyapatite

crystal formation Impaired calcium binding to hydroxyapatite

Directly alter crystal growth


have also been noted in the effect of Mg and calcium on

the biosynthesis of PTH in vitro. Changes in calcium over

the range of 0 to 3.0 m M resulted in increased PTH synthe-

sis ( Hamilton et al., 1971 ; Lee and Roth, 1975 ), whereas

changes in Mg over the range of 0 to 1.7 mM had no effect.

The effect of Mg on PTH secretion appears to act

through the Ca 2 1 -sensing receptor, which mediates the

control of extracellular calcium on PTH secretion ( Brown

et al., 1993 ). Mg 2 1 was shown to bind to this receptor, but

with much less efficiency than Ca 2 1 (Hebert , 1996). Mg

may also regulate calcium transport into the cell through

other ion channels ( Miki et al., 1997 ). Acute changes in

the serum Mg concentration may therefore modulate PTH

secretion and should be considered in the evaluation of the

determination of serum PTH concentrations.

Mg Depletion and Parathyroid Gland

Function

While acute changes in extracellular Mg concentrations

will influence PTH secretion qualitatively similar to cal-

cium, it is clear that Mg deficiency markedly perturbs min-

eral homeostasis ( Rude et al., 1976 ; Rude, 1994 ; Rude and

Shils, 2006 ). Hypocalcemia is a prominent manifestation of

Mg deficiency in humans ( Rude et al., 1976 ; Rude, 1994 ;

Rude and Shils, 2006 ), as well as in most other species

( Shils, 1980 ; Anast and Forte, 1983 ; Rude and Shils, 2006 ).

In humans, Mg deficiency must become moderate to severe

before symptomatic hypocalcemia develops. A positive cor-

relation has been found between serum Mg and calcium

concentrations in hypocalcemic hypomagnesemic patients

( Rude et al., 1976 ). Mg therapy alone restored serum cal-

cium concentrations to normal in these patients within days

( Rude et al., 1976 ). Calcium and/or vitamin D therapy will

not correct the hypocalcemia ( Rude et al., 1976 ; Rude,

1994 ). Even mild degrees of Mg depletion, however, may

result in a significant fall in the serum calcium concentra-

tion, as demonstrated in experimental human Mg depletion

( Fatemi et al., 1991 ). One major factor resulting in the fall

in serum calcium is impaired parathyroid gland function.

Low Mg in the media of parathyroid cell cultures impairs

PTH release in response to a low media calcium concentra-

tion ( Targovnik et al., 1971 ). Determination of serum PTH

concentrations in hypocalcemic hypomagnesemic patients

has shown heterogeneous results. The majority of patients

have low or normal serum PTH levels ( Anast et al., 1972 ;

Suh et al., 1973 ; Chase and Slatopolsky, 1974 ; Rude et al.,

1976 ; Rude et al., 1978 ). Normal serum PTH concentra-

tions are thought to be inappropriately low in the presence

of hypocalcemia. Therefore, a state of hypoparathyroidism

exists in most hypocalcemic Mg-deficient patients. Some

patients, however, have elevated levels of PTH in the serum

( Rude et al., 1976, 1978 ; Allgrove et al., 1984). The admin-

istration of Mg will result in an immediate rise in the serum

PTH concentration regardless of the basal PTH level ( Anast

et al., 1972 ; Rude et al., 1976 , Rude et al., 1978 ). As shown

in ( Fig. 7 ), 10 mEq of Mg administered intravenously over 1

minute caused an immediate marked rise in serum PTH in

patients with low, normal, or elevated basal serum PTH con-

centrations. This is distinctly different from the effect of an

Mg injection in normal subjects where, as discussed earlier,

Mg will cause an inhibition of PTH secretion ( Cholst et al.,

1984 ; Fatemi et al., 1991 ). The serum PTH concentration

will gradually fall to normal within several days of therapy

with return of the serum calcium concentration to normal

( Anast et al., 1972 ; Rude et al., 1976 ; Rude et al., 1978 ).

The impairment in PTH secretion appears to occur early in

Mg depletion. Normal human subjects placed experimen-

tally on a low Mg diet for only 3 weeks showed similar but

not as marked changes in the serum PTH concentrations

( Fatemi et al., 1991 ) in which there was a fall in both serum

calcium and PTH concentrations in 20 of 26 subjects at the

end of the dietary Mg deprivation period. The administration

of intravenous Mg at the end of this Mg depletion period

resulted in a significant rise in the serum PTH concentra-

tion qualitatively similar to that observed in hypocalcemic

Mg, 10 mEq Intravenous

Se

rum

Ca

mg

/dl

Se

rum

Mg

mg

/dl

Se

rum

IP

TH

pg

/ml

10

9

8

7

6

4

3

2

1

3600

3200

2800

1500

1000

500

Minutes

Undetectable

25 0 11 12 13 15 115

FIGURE 7 The effect of an IV injection of 10 mEq Mg on serum con-

centrations of calcium, magnesium, and immunoreactive parathyroid

hormone (IPTH) in hypocalcemic magnesium-deficient patients with

undetectable ( • ), normal (O), or elevated ( ∆ ) levels of IPTH. Shaded

areas represent the range of normal of each assay. The broken line for the

IPTH assay represents the level of detectability. The magnesium injection

resulted in a marked rise in PTH secretion within 1 minute in all three

patients ( Rude et al., 1978 ).


Mg-depleted patients shown in Fig. 7 , whereas a similar

Mg injection suppressed PTH secretion prior to the low

Mg diet. In this study, as with hypocalcemia hypomagne-

semic patients, some subjects had elevations in the serum

PTH concentration. The heterogeneous serum PTH val-

ues may be explained on the severity of Mg depletion. As

the serum Mg concentration falls, the parathyroid gland

will react normally with an increase in PTH secretion. As

intracellular Mg depletion develops, however, the ability of

the parathyroid to secrete PTH is impaired, resulting in a

fall in serum PTH levels with a resultant fall in the serum

calcium concentration. This concept is supported by the

observation that the change in serum PTH in experimen-

tal human Mg depletion is correlated positively with the

fall in red blood cell intracellular-free Mg 2 1 ( Fatemi et al.,

1991 ). A slight fall in red blood cell Mg 2 1 resulted in an

increase in PTH. However, a greater decrease in red blood

cell Mg 2 1 correlated with a progressive fall in serum PTH

concentrations.

It is conceivable that either PTH synthesis and/or

PTH secretion may be affected. However, as the in vitro

biosynthesis of PTH requires approximately 45 minutes

( Hamilton et al., 1971 ), the immediate rise in PTH fol-

lowing the administration of intravenous magnesium to

Mg-deficient patients strongly suggests that the defect is in

PTH secretion.

Mg Depletion and Parathyroid Hormone

Action

The previous discussion strongly supports the notion that

impairment in the secretion of PTH in Mg deficiency is a

major contributing factor in the hypocalcemia. However,

the presence of normal or elevated serum concentrations of

PTH in the face of hypocalcemia ( Rude et al., 1976 ; Rude

et al., 1978 ; Rude, 1994 ) suggests that there may also be

end-organ resistance to PTH action. In hypocalcemic

Mg-deficient patients treated with Mg, the serum calcium

concentration does not rise appreciably within the first 24

hours, despite elevated serum PTH concentrations ( Rude

et al., 1976 ; Rude, 1994 ), which also suggests skeletal

resistance to PTH because exogenous PTH administered to

hypoparathyroid patients causes a rise in the serum calcium

within 24 hours ( Bethune et al., 1968 ). Clinical studies

have reported resistance to exogenous PTH in hypocal-

cemic Mg-deficient patients ( Estep et al., 1969 ; Woodard

et al., 1972 ; Rude et al., 1976 ; Rude, 1994 ; Mihara et al .,

1995 ; Klein and Herndon, 1998 ; Sahota et al ., 2006 ). In

one study, parathyroid hormone did not result in elevation

in the serum calcium concentration or urinary hydroxypro-

line excretion in hypocalcemic hypomagnesemic patients

as shown in ( Fig. 8 ) ( Estep et al., 1969 ). Following Mg

repletion, however, a clear response to PTH was observed.

PTH has also been shown to have a reduced calcemic effect

in Mg-deficient animals ( MacManus et al., 1971 ; Levi et

al., 1974 ; Forbes and Parker, 1980 ). The ability of PTH to

resorb bone in vitro is also diminished greatly in the pres-

ence of low media Mg ( Raisz and Niemann, 1969 ). In one

study of isolated perfused femur in the dog, the ability of

PTH to simulate an increase in the venous cyclic AMP was

impaired during perfusion with low Mg fluid, suggesting

skeletal PTH resistance ( Freitag et al., 1979 ). Not all

studies have shown skeletal resistance to PTH, however

( Salet et al., 1966 ; Stromme et al., 1969 ; Suh et al., 1973 ,

Chase and Slatopolsky, 1974 ). It appears likely that skel-

etal PTH resistance may be observed in patients with more

severe degrees of Mg depletion. Patients in whom a nor-

mal calcemic response to PTH was demonstrated were in

subjects who had been on recent Mg therapy ( Salet et al.,

1966 ; Stromme et al., 1969 ; Suh et al., 1973 , Chase and

Slatopolsky, 1974 ). Patients who have been found to be

resistant to PTH have, in general, not had prior Mg admin-

istration ( Estep et al., 1969 ; Woodard et al., 1972 ; Rude

12

60

100

90

80

70

40

20

10

8

6

Control PTHControl PTHControl PTH

SerumCamg

100ml

%TRP

Urinehypro

mg

day

* mqSo4 24248 mEq I.V. daily 3 3

FIGURE 8 Mean and standard deviations of serum calcium concentration and urinary hydroxyproline and phosphate excretion in hypocalcemic

magnesium-deficient patients before ( • ) and after ( m ) 3 days of parenteral magnesium therapy ( Estep et al., 1969 ).


et al., 1976 ; Rude, 1994 ). Consistent with this notion is

that in the Mg-depleted rat, normal responses to PTH were

observed when the serum Mg concentration was 0.95 mg/dl

( Hahn et al., 1972 ); however, in another study, rats with

a mean serum Mg of 0.46 mg/dl were refractory to PTH

( MacManus et al., 1971 ). In addition, a longitudinal study

of Mg deficiency in dogs demonstrated a progressive

decline in responsiveness to PTH with increasing degrees

of Mg depletion ( Levi et al., 1974 ).

Calcium release from the skeleton also appears to be

dependent on physicochemical processes as well as cellu-

lar activity ( Pak and Diller, 1969 ; MacManus and Heaton,

1970 ). Low Mg will result in a decrease in calcium release

from bone ( Pak and Diller, 1969 ; MacManus and Heaton,

1970 ) and may be another mechanism for hypocalcemia in

Mg deficiency.

The renal response to PTH has also been assessed by

determining the urinary excretion of cyclic AMP and/or

phosphate ( Figs. 8 and 9 ) in response to exogenous PTH.

In some patients, a normal effect of PTH on urinary phos-

phate and cyclic AMP excretion has been noted ( Anast

et al., 1972 ; Suh et al., 1973 ; Chase and Slatopolsky,

1974 ). In general, these were the same subjects in which

a normal calcemic effect was also seen ( Anast et al.,

1972 ; Suh et al., 1973 ; Chase and Slatopolsky, 1974 ). In

other studies, with more severely Mg-depleted patients,

an impaired response to PTH has been observed ( Estep

et al., 1969 ; Rude et al., 1976 ; Medalle and Waterhouse,

1973 ; Rude, 1994 ; Mihara et al ., 1995 ). A decrease in uri-

nary cyclic AMP excretion in response to PTH has also

been described in the Mg-deficient dog and rat ( Levi et al.,

1974 ; Forbes and Parker, 1980 ).

Mechanism of Impaired Mineral

Homeostasis in Mg Depletion

The mechanism for impaired PTH secretion and action

in Mg deficiency remains unclear. It has been suggested

that there may be a defect in the second messenger sys-

tems in Mg depletion. PTH is thought to exert its biologic

effects through the intermediary action of cyclic AMP

( Bitensky et al., 1973 ; Neer, 1995 ). Adenylate cyclase

has been universally found to require Mg for cyclic AMP

generation, both as a component of the substrate (Mg-ATP)

and as an obligatory activator of enzyme activity ( Northup

et al., 1982 ). There appears to be two Mg 2 1 -binding sites

within the adenylate cyclase complex: one resides on the

catalytic subunit and the other on the guanine nucleotide

regulatory protein, Ns ( Cech et al., 1980 ; Maguire, 1984 ).

The requisite role that Mg 2 1 plays in adenylate cyclase

function suggests that factors that would limit the availabil-

ity of Mg 2 1 to this enzyme could have significant effects

on the cyclic nucleotide metabolism of a cell and hence

overall cellular function. It is clear that some patients with

severe Mg deficiency have a reduced urinary excretion of

cyclic AMP in response to exogenously administered PTH

( Rude et al., 1976 ). In addition, PTH was shown to have a

blunted effect in causing a rise in cyclic AMP from isolated

perfused tibiae in Mg-deficient dogs ( Freitag et al., 1979 ).

These observations correspond well with the impaired cal-

cemic and phosphaturic effects of PTH in Mg-deficient

patients and animals as discussed earlier.

While Mg 2 1 is stimulatory for adenylate cyclase, Ca 2 1

may inhibit or activate enzyme activity ( Sunahara et al.,

1996 ). Nine isoforms of adenylate cyclase have been iden-

tified whose activities are modulated by both Mg 2 1 and

Ca 2 1 ( Sunahara et al., 1996 ). In plasma membranes from

parathyroid, renal cortex, and bone cells, Ca 2 1 will com-

petitively inhibit Mg 2 1 -activated adenylate cyclase activity

( Rude, 1983 ; Rude, 1985 ; Oldham et al., 1984 ). In parathy-

roid plasma membranes, at a Mg 2 1 concentration of 4 m M,

Ca 2 1 was found to inhibit adenylate cyclase in a bimodal

pattern described in terms of two calcium inhibition con-

stants with K i values of 1–2 and 200–400 µ M ( Oldham

et al., 1984 ). At a lower Mg 2 1 concentration (0.5 m M ) the

only adenylate cyclase activity expressed was that inhibit-

able by the high-affinity Ca 2 1 -binding site. With increasing

Mg concentrations, the fraction of total adenylate cyclase

activity subject to high-affinity calcium inhibition became

progressively less. Thus, the ambient Mg 2 1 concentrations

can markedly affect the susceptibility of this enzyme to

the inhibitory effects of Ca 2 1 . Total intracellular calcium

has been observed to rise during Mg depletion ( George

and Heaton, 1975 ; Ryan and Ryan, 1979 ). Mg is not only

important for the operation of Mg 2 1 , Ca 2 1 -dependent

ATPase, but may also be countertransported during the

uptake and release of calcium through calcium channels

( Romani and Scarpa, 1992 ; Romani, 2006 ). The combina-

tion of higher intracellular Ca 2 1 and increased sensitivity

400

300

200

100

008–09 09–10

PTE200 ui.v.

Time (hours)

Urin

ary

cyclic

AM

P(

mo

les/g

cre

atin

ine

)

10–11 11–12

µ

FIGURE 9 The effect of an IV injection of 200 units of parathyroid

extract on the excretion of urinary cyclic AMP in a magnesium-deficient

patient before ( • --- • ) and after ( • — • ) 4 days of magnesium therapy.

Urine was collected for four consecutive 1-hour periods, two before and

two after the PTE injection. Although Mg-deficient, the patient had a

minimal rise in urinary cyclic AMP in response to PTH, but following

Mg therapy the response was normal ( Rude et al., 1976 ).


to Ca 2 1 inhibition because of Mg depletion could explain

the defective PTH secretion in Mg deficiency. An increase

in the release of intracellular Ca 2 1 via the phosphoinosi-

tol system is also possible, as discussed later. A similar

relationship between Mg 2 1 and Ca 2 1 was described for

adenylate cyclase obtained from bone ( Rude, 1985 ). Ca 2 1

caused a competitive inhibition of Mg 2 1 -activated skeletal

adenylate cyclase with a high-affinity Ca 2 1 -binding site

with a K i Ca of 1–2 µ M . Lowering the Mg 2 1 concentration

increased overall Ca 2 1 inhibition. Thus, a fall in the intra-

cellular Mg 2 1 concentration would render the adenyl-

ate cyclase enzyme more susceptible to inhibition by the

prevailing intracellular Ca 2 1 concentrations and may be a

mechanism by which both PTH secretion and PTH end-

organ action are compared in Mg deficiency.

Adenylate cyclase is a widely distributed enzyme in the

body, and if the hypothesis just given were true, the secre-

tion and action of other hormones mediated by adenylate

cyclase might also exhibit impaired activity in Mg defi-

ciency. This has not been found to be true, as the actions

of ACTH, TRH, GnRH, and glucagon are normal in Mg

depletion ( Cohan et al., 1982 ). Prior investigations have

suggested that Mg affinity for adenylate cyclase is higher

(lower K a Mg) in liver, adrenal, and pituitary than in para-

thyroid (for reviews, see Rude and Oldham, 1985 ; Rude,

1994 ). In one study, investigation of K a Mg and K i Ca in

tissues from one species (guinea pig) demonstrated that

under agonist stimulation, the K a Mg from liver is less

than thyroid less than kidney, which equals bone, and the

KiCa2 1 for liver greater than renal greater than kidney also

equals bone ( Rude and Oldham, 1985 ). These data suggest

that adenylate cyclase regulation by divalent cations varies

from tissue to tissue and may explain the greater propen-

sity for disturbed mineral homeostasis in Mg deficiency.

Although cyclic AMP is an important mediator of PTH

action, current studies do not suggest an important role in

mediating Ca 2 1 -regulated PTH secretion ( Brown, 1991 ;

Dunlay and Hruska, 1990 ). PTH has been shown to activate

the phospholipase C second messenger system ( Dunlay and

Hruska, 1990 ). PTH activation of phospholipase C leads

to the hydrolysis of phosphatidylinositol 4,5-bisphosphate

to inositol-1,4,5-triphosphate (IP 3 ) and diacylglycerol. IP 3

binds to specific receptors on intracellular organelles (endo-

plasmic reticulum, calciosomes), leading to an acute tran-

sient rise in cytosolic Ca 2 1 with a subsequent activation

of calmodulin-dependent protein kinases. Diacylglycerol

activates protein kinase C. Mg depletion could perturb this

system via several mechanisms. First, an Mg 2 1 -dependent

guanine nucleotide-regulating protein is also involved in the

activation of phospholipase C ( Babich et al., 1989 ; Litosch,

1991 ). Mg 2 1 has also been shown to be a noncompetitive

inhibitor of IP 3 -induced Ca 2 1 release ( Volpe et al., 1990 ).

A reduction of Mg 2 1 from 300 to 30 µ M increased Ca 2 1

release in response to IP 3 by two- to threefold in mitochon-

drial membranes obtained from canine cerebellum ( Volpe

et al., 1990 ). The Mg concentration required for a half-

maximal inhibition of IP 3 -induced Ca 2 1 release was 70 µ U.

In these same studies, Mg 2 1 was also found to inhibit IP 3

binding to its receptor. Mg, at a concentration of 500 µ M ,

decreased maximal IP 3 binding threefold (IC 50 200 µ M )

( Volpe et al., 1990 ). These Mg 2 1 concentrations are well

within the estimated physiologic intracellular range (200–

500 µ M ) and therefore Mg 2 1 may be an important physio-

logical regulator of the phospholipase C second messenger

system. These effects may be related to Mg deficiency-

induced activation of the α -subunit of the Ca-sensing

receptor ( Quitterer et al., 2001 ; Vetter and Lohse, 2002 ).

The effect of Mg depletion on cellular function in terms

of the second messenger systems is most complex, poten-

tially involving substrate availability, G protein activity,

release and sensitivity to intracellular Ca 2 1 , and phospho-

lipid metabolism.

Mg Depletion and Vitamin D Metabolism

and Action

Mg may also be important in vitamin D metabolism and/

or action. Patients with hypoparathyroidism, malabsorp-

tion syndromes, and rickets have been reported to be

resistant to therapeutic doses of vitamin D until Mg was

administered simultaneously (for review, see Rude, 1994 ).

Patients with hypocalcemia and Mg deficiency have also

been reported to be resistant to pharmacological doses of

vitamin D ( Medalle et al., 1976 ; Leicht et al., 1990 ), 1 α

hydroxyvitamin D ( Ralston et al., 1983 ; Selby et al., 1984 )

and 1,25-dihydroxyvitamin D ( Graber and Schulman,

1986 ). Similarly, an impaired calcemic response to vita-

min D has been found in Mg-deficient rats ( Lifshitz et al.,

1967 ), lambs (McAleese and Forbes, 1959), and calves

( Smith, 1958 ).

The exact nature of altered vitamin D metabolism

and/or action in Mg deficiency is unclear. Intestinal cal-

cium transport in animal models of Mg deficiency has

been found to be reduced in some ( Higuchi and Lukert,

1974 ) but not all ( Coburn et al., 1975 ) studies. Calcium

malabsorption was associated with low serum levels of

25-hydroxyvitamin D in one study ( Lifshitz et al., 1967 ),

but not in another ( Coburn et al., 1975 ), suggesting that

Mg deficiency may impair intestinal calcium absorption

by more than one mechanism. Patients with Mg deficiency

and hypocalcemia frequently have low serum concentra-

tions of 25-hydroxyvitamin D ( Rude et al., 1985 ; Fuss

et al., 1989 ) and therefore nutritional vitamin D deficiency

may be one factor. Therapy with vitamin D, however,

results in high serum levels of 25-hydroxyvitamin D with-

out correction of the hypocalcemia ( Medalle et al., 1976 ),

suggesting that the vitamin D nutrition is not the major

reason. In addition, conversion of radiolabeled vitamin D

to 25-hydroxyvitamin D was found to be normal in three


Mg-deficient patients ( Lukert, 1980 ). Serum concentra-

tions of 1,25-dihydroxyvitamin D have also been found

to be low or low normal in most hypocalcemic Mg-defi-

cient patients ( Rude et al., 1985 ; Fuss et al., 1989 ; Leicht

et al., 1992 ; Rude and Shils, 2006 ). Mg-deficient diabetic

children, when given a low calcium diet, did not exhibit

the expected normal rise in serum 1,25-dihydroxyvitamin

D or PTH ( Saggese et al., 1988 ); the response returned

to normal following Mg therapy ( Saggese et al., 1991 ).

Because PTH is a major trophic for 1,25-dihydroxyvi-

tamin D formation, the low serum PTH concentrations

could explain the low 1,25-dihydroxyvitamin D levels. In

support of this is the finding that some hypocalcemic Mg-

deficient patients treated with Mg have a rise in serum 1,25-

dihydroxyvitamin D to high normal or to frankly elevated

levels, as shown in ( Fig. 10 ) ( Rude et al., 1985 ). Most

patients, however, do not have a significant rise within 1

week after institution of Mg therapy, despite a rise in serum

PTH and normalization of the serum calcium concentration

( Fig. 10 ) ( Rude et al., 1985 ). These data suggest that Mg

deficiency in humans also impairs the ability of the kidney

to synthesize 1,25-dihydroxyvitamin D. This is supported

by the observation that the ability of exogenous admin-

istration of 1–34 human PTH to normal subjects after 3

weeks of experimental Mg depletion resulted in a signifi-

cantly lower rise in serum 1,25-dihydroxyvitamin D con-

centrations than before institution of the diet ( Fatemi et al.,

1991 ). It appears, therefore, that the renal synthesis of 1,25-

dihydroxyvitamin D is sensitive to Mg depletion. Although

Mg is known to support 25-hydroxy-1 α -hydroxylase in

vitro ( Fisco and Traba, 1992 ), the exact Mg requirement

for this enzymatic process is not known. Bone-specific

binding sites for 1,2-dihydroxyvitamin D have also been

described to be reduced in Mg deficiency ( Risco and

Traba, 2004 ).

The association of Mg deficiency with impaired vita-

min D metabolism and action therefore may be because of

several factors, including vitamin D deficiency ( Rude et al.,

1985 ; Carpenter , 1988; Fuss et al., 1989 ; Leicht and Biro,

1992) and a decrease in PTH secretion ( Anast et al., 1972 ;

Suh et al., 1973 ; Chase and Slatopolsky, 1974 ; Rude et al.,

1976 ; Rude et al., 1978 ), as well as a direct effect of Mg

depletion on the ability of the kidney to synthesize 1,25-

dihydroxyvitamin D ( Rude et al., 1985 ; Fuss et al., 1989 ;

Fatemi et al., 1991 ). Osteoporotic patients with a blunted

response to PTH exhibit Mg deficiency ( Sahota et al.,

2006 ). In addition, Mg deficiency may directly impair

intestinal calcium absorption ( Higuchi and Lukert, 1974 ;

Rude et al., 1976 ; Rude et al., 1985 ). Skeletal resistance to

vitamin D and its metabolites may also play an important

role ( Lifshitz et al., 1967 ; Ralston et al., 1983 ; Selby et al.,

1984 ; Graber and Schulman, 1986 ). It is clear, however,

that the restoration of normal serum 1,25-dihydroxyvitamin

D concentrations is not required for normalization of the

serum calcium level ( Fig. 9 ). Most Mg-deficient patients

who receive Mg therapy exhibit an immediate rise in PTH,

followed by normalization of the serum calcium prior to

any change in serum 1,25-dihydroxyvitamin D concen-

trations ( Rude et al., 1985 ; Fuss et al., 1989 ). An overall

view of the effect of Mg depletion on calcium metabolism

is shown in Fig. 11 .

MAGNESIUM DEPLETION: SKELETAL

GROWTH AND OSTEOPOROSIS

Women with postmenopausal osteoporosis have decreased

nutrition markers, suggesting that osteoporosis is asso-

ciated with nutritional deficiencies ( Rico et al., 1993 ;

Ames, 2006 ). A low calcium intake is one of these nutri-

tional factors ( Rico et al., 1993 ) and predicts bone mineral

density and fracture rate ( Dawson-Hughes et al. , 1990 ;

Chapuy et al ., 1992 ). A large segment of our popula-

tion also has low dietary Mg intake ( Morgan et al., 1985 ;

Marier, 1986 ; Cleveland et al., 1994). Mg deficiency, when

severe, will disturb calcium homeostasis markedly, result-

ing in impaired PTH secretion and PTH end organ resis-

tance, leading to hypocalcemia ( Rude, 1998 ). Mg exists

in macronutrient quantities in bone, and long-term, mild-

to-moderate dietary Mg deficiency has been implicated

as a risk factor for osteoporosis ( Sojka and Weaver, 1995 ;

Cohen and Kitzes, 1981 ).

10

9

8

7

6

80

70

60

50

40

30

20

10

0

Before Mg therapy

Se

rum

1.2

5(O

H) 2

-D p

g/m

lS

eru

m C

a m

g/d

l

After Mg therapy

Undetectable

(116)(99)

FIGURE 10 Serum concentrations of calcium and 1,25-diydroxyvi-

tamin D in hypocalcemic magnesium-deficient patients before and after

5–8 days of parenteral magnesium therapy. The broken line represents the

upper and lower limits of normal for serum 1,25-dihydroxyvitamin D and

the lower limit of normal for the serum calcium ( Rude et al., 1985 ).


Epidemiological Studies

Epidemiological studies have provided a major link asso-

ciating dietary Mg inadequacy to osteoporosis. One cross-

sectional study assessed the effect of dietary nutrients on

appendicular (radius, ulna, and heel) bone mineral density

(BMD) in a large group of Japanese Americans living in

Hawaii ( Yano et al., 1985 ). In 1,208 males (ages 61–81),

whose mean Mg intake was 238 6 111 mg/day, no correla-

tion of Mg intake with BMD was observed at any site. In

a subgroup of 259 of these subjects who took Mg supple-

ments, however (mean Mg intake of 381 mg/day), BMD

was correlated positively with Mg intake at one or more

skeletal sites. In 912 females (ages 43–80) whose Mg

intake was 191 6 36 mg/day, a positive correlation with

BMD was also observed. In contrast to males, no correla-

tion with BMD was found in females who took Mg supple-

ments (total Mg intake of 321 mg/day). In a smaller study

of women ages 35–65 (17 premenopausal, mean Mg intake

243 6 44 mg/day; 67 postmenopausal, mean Mg intake

249 6 68 mg/day) in which BMD was measured in the

distal forearm, no cross-sectional correlation was observed

with Mg intake in either group ( Freudenheim et al., 1986 ).

Longitudinal observation over 4 years, however, dem-

onstrated that loss of bone mass was related inversely to

Mg intake in premenopausal women ( p , 0.05) and had

a similar trend in the postmenopausal group ( p , 0.085).

In another cross-sectional study, a positive correlation of

BMD of the forearm (but not femur or spine) was found in

a larger group of 89 premenopausal women (age 37.8 6 0.8;

Mg intake of 243 6 9 mg/day), but no similar correla-

tion was found in 71 recently menopausal women ages

58.9 6 0.9 (Mg intake of 253 6 11 mg/day) ( Angus et al.,

1988 ). In contrast, a study of 194 older postmenopausal

women (ages 69–97; mean Mg intake of 288 mg/day) dem-

onstrated a significant positive correlation with BMD of

the forearm ( Tranquilli et al., 1994 ).

Other studies have concentrated on BMD of the axial

skeleton. Sixty-six premenopausal women (ages 28–39)

whose Mg intake was 289 6 73 mg/day had a signifi-

cant relationship between dietary Mg intake and rate of

change of BMD of the lumbar spine and total body cal-

cium over a 1-year period ( Houtkooper et al., 1995 ). A

cross-sectional study that combined 175 premenopausal

and postmenopausal women ages 28–74 (mean Mg intake

was 262 6 70 mg/day) found no correlation with BMD

at the lumbar spine, femoral neck, or total body cal-

cium ( Michaelsson et al., 1995 ). In a study of 994 pre-

menopausal women ages 45–49, whose Mg intake was

311 6 85 mg/day, New et al. (1997) found a significant

correlation of BMD of the lumbar spine with Mg intake.

A significant difference was also observed in lumbar

spine BMD between the highest and the lowest quartiles

of dietary Mg intake. A report by this same group in a

study of 65 pre- and postmenopausal women ages 45–55

again found higher bone mass of the forearm (but not

femoral neck or hip) in subjects consuming a Mg intake

of 326 6 90 mg/day ( New et al., 2000 ). Women with a

high childhood intake of fruits (Mg and potassium) did

have higher femoral neck BMD than those on a lower fruit

intake, however. Another cross-sectional study assessed

Mg intake in older males and females (ages 69–97) (345

males and 562 females), as well as a 2-year longitudinal

study of a subset of these subjects (229 males and 399

females) ( Tucker et al., 1999 ). In the cross-sectional analy-

sis in males, Mg intake (300 6 110 mg/day) was correlated

Effect of parathyroid hormone

1,25-OH2 vitamin D

Effect of parathyroid hormone

Parathyroid hormone

Parathyroid gland

PlasmaCa

Ca

Ca

Ca

Ca

Ca

Ca

Ca11

FIGURE 11 Disturbance of calcium metabolism during magnesium deficiency. Hypocalcemia is caused by a decrease in PTH secretion, as well as

renal and skeletal resistance to the action of PTH. Low serum concentrations of 1,25-dihydroxyvitamin D may result in reduced intestinal calcium

absorption (Rude and Oldham, 1990).


with BMD of the radius and hip. In the 4-year longitudi-

nal study of these subjects, a positive inverse relationship

between bone loss of the hip and Mg intake was observed.

A positive cross-sectional correlation of BMD of the hip

was also observed in females (Mg intake of 288 6 106 mg/

day), but not in the longitudinal assessment. Dietary Mg

intake in a large population of non-Hispanic white males

and females from the NHANES III database was also

found by multiple regression analysis to predict BMD at

several sites in the proximal femur ( Carpenter et al., 2000 ).

Most recently, Mg intake was significantly correlated with

whole body and hip BMD in elderly white females and

males ( Ryder et al ., 2005 ).

In a study of younger individuals, the effect of dietary

Mg intake of preadolescent girls (ages 9–11) on bone mass/

quality in these young women was evaluated at age 18–19

( Wang et al., 1999 ). Ultrasound determination of bone

mass of the calcaneus in 35 African American women (Mg

intake 237 6 83 mg/day) and in 26 Caucasian women (Mg

intake 240 6 61 mg/day) was performed. Mg intake was

related positively to quantitative ultrasound properties of

bone, suggesting that this nutrient was important in skeletal

growth and development. Bounds et al . (2005) reported that

bone mineral content was significantly related to Mg intake

at age 8, including longitudinal intake from age 2 to 8.

In summary, these epidemiological studies link dietary

Mg intake to bone mass. Exceptions appear to include

women in the early postmenopausal period in which the

effect of acute sex steroid deficiency may mask the effect

of dietary factors such as Mg. In addition, diets deplete in

Mg are usually deficient in other nutrients, which affect

bone mass as well. Therefore, further investigations are

needed to provide a firm relationship of dietary Mg inad-

equacy with osteoporosis.

Bone Turnover

In two of the epidemiological studies cited earlier, markers

of bone turnover were determined. In one, where no cor-

relation was found between BMD and dietary Mg intake,

serum osteocalcin did not correlate with Mg (or any other

nutrient) intake ( Michaelsson et al., 1995 ). New et al.

(2000) also found that serum osteocalcin was not associ-

ated with the dietary intake of Mg or other nutrients. Mg

intake, however, was significantly negatively correlated

with the urinary excretion of pyridinoline and deoxypyridi-

noline, suggesting that a low Mg diet was associated with

increased bone resorption ( New et al., 2000 ).

The affect of short-term administration of Mg on bone

turnover in young normal subjects has been conflict-

ing. Magnesium, 360 mg per day, was administered for

30 days to 12 normal males age 27 (mean dietary intake

prior to supplementation was 312 mg/day) and markers of

bone formation (serum osteocalcin and C terminus of type

I procollagen) and bone resorption (type I collagen telopep-

tide) were compared with 12 age-matched controls ( Dimai

et al., 1998 ). Markers of both formation and resorption

were suppressed significantly however, only during the first

5–10 days of the study. A similar trial of 26 females ages

20–28 in a double-blind, placebo-controlled, randomized

crossover design has been reported ( Doyle et al., 1999 ).

Magnesium, 240 mg/day, or placebo was administered for

28 days (mean dietary Mg intake was 271 mg/day prior to

and during the study). No effect of Mg supplementation

was observed on serum osteocalcin, bone-specific alkaline

phosphatase, or urinary pyridinoline and deoxypyridinoline

excretion.

Mg Status in Osteoporosis

Significant correlation between serum Mg and bone

metabolism has been observed. Gur et al . (2002) demon-

strated lower serum values of Mg in 70 osteoporotic sub-

jects compared to 30 nonosteoporotic postmenopausal

women. In a similar study involving 20 perimenopausal

and 53 postmenopausal women, women with severe

osteoporosis had significantly lower ionized Mg levels

(Brodowski, 2002) . This was also confirmed in another

study ( Saito et al ., 2004 ). In a study of 168 patients with

Crohn’s disease, higher serum Mg predicted higher BMD

at the femur (Habtezion et al ., 2002). A study of a fam-

ily with primary hypomagnesemia owing to renal Mg

wasting demonstrated significant reductions in serum and

lymphocyte Mg concentrations; affected members had

significantly reduced BMD at the lumbar spine and proxi-

mal femur ( Kantorovich et al. , 2002 ). Mg is principally an

intracellular cation, therefore serum Mg concentration may

not reflect Mg status. Researchers have employed Mg tol-

erance testing, red blood cell Mg, and skeletal Mg content.

A small group of 15 osteoporotic subjects (10 female, 5

male) ages 70–85 (the presence or absence of osteoporosis

was determined by radiographic features) was compared

to 10 control nonosteoporotic subjects ( Cohen and Kitzes,

1983 ). Both groups had normal serum Mg concentrations,

which were not significantly different from each other. The

Mg tolerance test, however, revealed a significantly greater

retention in the osteoporotic patients (38%) as compared

to 10% in the control subjects, suggesting Mg deficiency.

In a second study by this group, 12 younger women ages

55–65 with osteoporosis (as determined by x-ray) had sig-

nificantly lower serum Mg concentrations than 10 control

subjects; however, no difference in the Mg tolerance test

was observed ( Cohen et al., 1983 ). Red blood cell Mg

was found to be significantly lower in 10 postmenopausal

women who had at least one vertebral fracture as compared

to 10 subjects with degenerative osteoarthritis; however,

no difference in plasma Mg was found ( Reginster et al.,

1985 ). In a second report, 10 postmenopausal women ages


68.9 6 9 with vertebral crush fracture were compared to 10

nonosteoporotic women ages 67.2 6 6 years ( Reginster,

1989 ). In comparison to the 10 controls, the osteoporotic

subjects had a significantly lower serum Mg, but no differ-

ence was noted in red blood cell Mg.

The majority of body Mg (50–60%) resides in the skel-

eton, and skeletal Mg reflects Mg status. In the two stud-

ies cited earlier in which an Mg deficit was suggested

by either Mg tolerance testing or low serum Mg concen-

tration, the Mg content of iliac crest trabecular bone was

reduced significantly in osteoporotic patients ( Cohen and

Kitzes, 1983 ; Cohen et al., 1983 ). Two additional studies

also found a lower bone Mg content in elderly osteoporotic

patients ( Manicourt et al., 1981 ; Milachowski et al., 1981 ).

However, no difference in bone Mg content between osteo-

porotic subjects and bone obtained from cadavers was

found ( Reginster, 1989 ). Another study found no differ-

ence between patients with osteoporosis and control sub-

jects in cortical bone ( Basle et al., 1990 ), while two studies

reported higher bone Mg content in osteoporosis ( Basle

et al., 1990 ; Burnell et al., 1982 ).

In summary, Mg status has been assessed in very few

osteoporotic patients. Low serum and red blood cell Mg

concentrations, as well as a high retention of parenter-

ally administered Mg, suggest an Mg deficit; however,

these results are not consistent from one study to another.

Similarly, although a low skeletal Mg content has been

observed in some studies, others have found normal or

even high Mg content. Larger scale studies are needed.

Effect of Mg Therapy in Osteoporosis

The effect of dietary Mg supplementation on bone mass

in patients with osteoporosis has not been studied exten-

sively. Administration of 600 mg of Mg per day to 19

patients over 6–12 months ( Abraham, 1991 ) was reported

to increase BMD of the calcaneus (11%) compared to

a 0.7% rise in 7 control subjects. All subjects were post-

menopausal (ages 42–75) and on sex steroid replacement

therapy. Subjects who received Mg also received 500 mg

of calcium per day, as well as many other dietary supple-

ments, making if difficult to conclude if Mg alone was the

sole reason for the increase in bone mass. In a retrospec-

tive study, Mg (200 mg per day) given to 6 postmenopausal

women (mean age 59) was observed to have a small non-

significant 1.6% rise in bone density of the lumbar spine;

no change was seen in the femur ( Eisinger and Clairet,

1993 ). Stendig-Lindberg et al. (1993) conducted a 2-year

trial in which 31 postmenopausal osteoporotic women

were administered 250 mg Mg per day, increasing to a

maximum of 750 mg per day for 6 months depending on

tolerance. All subjects were given 250 mg Mg per day from

months 6 to 24. Twenty-three age-matched subjects served

as controls. At 1 year there was a significant 2.8% increase

in bone density of the distal radius. Twenty-two of the 31

subjects had an increase in bone density while 5 did not

change. Three subjects that showed a decrease in bone den-

sity had primary hyperparathyroidism and one underwent a

thyroidectomy. No significant effect of Mg supplementa-

tion was shown at 2 years, although only 10 subjects com-

pleted the trial. In a small uncontrolled trial, a significant

increase in bone density of the proximal femur and lumbar

spine in celiac sprue patients who received approximately

575 mg Mg per day for 2 years was reported ( Rude and

Olerich, 1996 ). These subjects had demonstrated evidence

of reduced free Mg in red blood cells and peripheral lym-

phocytes. Recently, Carpenter et al . (2006) administered

300 mg Mg to girls ages 8–14 over 1 year and noted a sig-

nificantly greater increase in hip bone mineral content in

the Mg-treated group.

In summary, the effect of Mg supplements on bone

mass has generally led to an increase in bone mineral

density, although study design limits useful information.

Larger, long-term, placebo-controlled, double-blind inves-

tigations are required.

Osteoporosis in Patients at Risk for Mg

Defi ciency

Osteoporosis may occur with greater than usual frequency

in certain populations in which Mg depletion is also com-

mon. These may include diabetes mellitus ( Levin et al.,

1976 ; McNair et al., 1979 , McNair et al., 1981 ; Hui et al.,

1985 ; Saggese et al., 1988 ; Krakauer et al., 1995 ), chronic

alcoholism ( Bikle et al., 1985 ; Lindholm et al., 1991 ;

Peris et al., 1992 ), and malabsorption syndromes ( Molteni

et al., 1990 ; Mora et al., 1993 ). Changes in bone and min-

eral metabolism in patients with diabetes mellitus and alco-

holism are surprisingly similar to those in Mg depletion as

discussed earlier. Serum PTH and/or 1,25(OH) 2 D concen-

trations have been found to be reduced in both human and

animal studies ( McNair et al., 1979 ; Hough et al., 1981 ;

Imura et al., 1985 ; Ishida et al., 1985 ; Nyomba et al., 1986 ;

Saggese et al., 1988 ; Verhaeghe et al., 1990 ). A prospec-

tive study of pregnant diabetic women demonstrated a fall

in serum 1,25(OH) 2 D during pregnancy rather than the

expected rise observed in normal women ( Kuoppala, 1988 ).

These subjects were also found to have reduced serum Mg

concentrations. Diabetic children, with reduced serum Mg

and calcium concentrations and low bone mineral content,

were shown to have an impaired rise in serum PTH and

1,25(OH) 2 D in response to a low calcium diet; this defect

normalized following Mg repletion ( Saggese et al., 1988 ,

Saggese et al., 1991 ). Similar observations were found in

the diabetic rat ( Welsh and Weaver, 1988 ). Duodenal cal-

cium absorption has also been reported to be low in dia-

betic rats ( Nyomba et al., 1989 ; Verhaeghe et al., 1990 ).

The calcium malabsorption may be because of low serum


1,25(OH) 2 D, as duodenal calbindin D 9K has been found to

be reduced ( Nyomba et al., 1989 ; Verhaeghe et al., 1990 ).

The reduction in bone mass in diabetes mellitus and alco-

holism also appears to be related to a decrease in bone forma-

tion, similar to what is observed in experimental Mg depletion

(see later; section 6) . A histomorphometric study of bone

has shown decreased bone formation, bone turnover, oste-

oid, and osteoblast number ( Tamayo et al., 1981 ; Goodman

and Hori, 1984 ; Verhaeghe et al., 1990 ; Hough et al., 1981 ;

Bouillon, 1991 ). Reduced bone turnover is supported by

the finding that serum osteocalcin, a marker of osteoblast

activity, is low in humans ( Pietschmann et al., 1988 ; Rico

et al., 1989 ) and in rats ( Ishida et al., 1988 ; Verhaeghe

et al., 1990 ). Patients with vitamin D insufficiency and osteo-

porosis were shown to have a blunted PTH response that was

related to Mg deficiency ( Sahota et al ., 2006 ).

Magnesium Depletion and Osteoporosis:

Experimental Animal Models

The effect of dietary Mg depletion on bone and mineral

homeostasis in animals has been studied since the 1940s.

Most studies have been performed in the rat. Dietary restric-

tion has usually been severe, ranging from 0.2 to 8 mg per

100 chow (normal 5 50–70 mg/100 g). A universal observa-

tion has been a decrease in growth of the whole body as well

as the skeleton ( Lai et al., 1975 ; McCoy et al., 1979 ; Mirra

et al., 1982 ; Carpenter et al., 1992 ; Boskey et al., 1992 ;

Kenny et al., 1994; Gruber et al., 1994 ). The epiphyseal and

diaphyseal growth plate is characterized by thinning and a

decrease in the number and organization of chrondrocytes

( Mirra et al., 1982 ). Osteoblastic bone formation has been

observed by quantitative histomorphometry to be reduced,

as shown in ( Fig. 12 ) ( Carpenter et al., 1992 ; Gruber et al.,

1994 , Rude et al., 1999 ). Serum and bone alkaline phospha-

tase ( Mirra et al., 1982 ; Loveless and Heaton, 1976 ; Lai et

al., 1975 ), serum and bone osteocalcin ( Boskey et al., 1992 ;

Carpenter et al., 1992 ; Creedon et al., 1999 ), and bone

osteocalcin mRNA ( Carpenter et al., 1992 ; Creedon et al.,

1999 ) have been reduced, suggesting a decrease in osteo-

blastic function. This is supported by an observed decrease

in collagen formation and sulfation of glycosaminoglycans

( Trowbridge and Seltzer, 1967 ). A decrease in tetracy-

cline labeling has also suggested impaired mineralization

( Carpenter et al., 1992 ; Jones et al., 1980 ). Dietary cal-

cium supplementation in the Mg-depleted rat will not pre-

vent loss of trabecular bone; indeed, a high calcium intake

suppressed bone formation even more ( Matsuzaki and

Miwa, 2006 ). Data on osteoclast function have been con-

flicting. A decrease in urinary hydroxyproline ( MacManus

and Heaton, 1969 ) and dexoypyridinoline ( Creedon

et al., 1999 ) has suggested a decrease in bone resorp-

tion; however, Rude et al. (1999) reported an increase in

the number and activity of osteoclasts in the Mg-deficient

rat, as shown in ( Fig. 13 ). Bone from Mg-deficient rats

has been described as brittle and fragile ( Lai et al., 1975 ;

Duckworth et al., 1940 ). Biomechanical testing has directly

demonstrated skeletal fragility in both rat and pig ( Boskey

et al., 1992 ; Kenny et al., 1992; Miller et al., 1965 ; Heroux

et al., 1975 ; Smith and Nisbet, 1968 ). Osteoporosis has

been observed to occur in dietary Mg depletion by 6 weeks

or longer ( Boskey et al., 1992 ; Carpenter et al., 1992 ; Rude

et al., 1999 ; Heroux et al., 1975 ; Smith and Nisbet, 1968 ),

as shown in ( Fig. 14 ). Bone implants into Mg-deficient

rats have also shown osteoporosis in the implanted bone

( Belanger et al., 1975 ; Schwartz and Reddi, 1979 ). A high

calcium diet will not affect this Mg deficiency-induced

reduction of bone mass ( Matsuzaki et al ., 2005 ). The effect

of higher than the recommended dietary Mg intake on

mineral metabolism in the rat has been reported ( Toba et

al., 2000 ). In this study, increasing dietary Mg from 48 to

118 mg/100 g chow resulted in a decrease in bone resorption

and an increase in bone strength in ovariectomized rats. No

loss of BMD was observed, suggesting a beneficial effect

of Mg in acute sex steroid deficiency.

These severe degrees of Mg deficiency are unlikely

to commonly occur in the human population. We have

recently reported less severe degrees of Mg restriction in

the rat (10%, 25%, and 50% of the recommended nutrient

requirement) and also observed detrimental effect on bone

( Rude et al ., 2004 ; Rude et al ., 2005 ; Rude et al ., 2006 )

including reduced trabecular bone, decreased bone forma-

tion, and increased bone resorption. The effect of an Mg

diet at 10% of the recommended nutrient requirement on

trabecular bone is demonstrated in Fig. 14 . These inad-

equate dietary Mg levels are present in segments of the

human population as discussed earlier and implicate Mg

deficiency as a possible risk factor for osteoporosis.

4 weeks

Nu

mb

er

of

oste

ob

lasts

/mm

tra

be

cu

lar

bo

ne

su

rfa

ce

16 weeks

2.5

2.0

1.5

1.0

0.5

0.0

*

* # .01p

FIGURE 12 After 16 weeks of magnesium deficiency in the rat (solid

bars), the osteoblast number was reduced significantly compared to con-

trols (open bars) ( Rude et al., 1999 ) * p , 0.01.


Possible Mechanisms for Mg Defi ciency-

Induced Osteoporosis

Several potential mechanisms may account for a decrease

in bone mass in Mg deficiency. Mg is mitogenic for bone

cell growth, which may directly result in a decrease in

bone formation ( Liu et al., 1988 ). Mg also affects crystal

formation; a lack of Mg results in a larger, more perfect

crystal, which may affect bone strength, as discussed later

( Cohen and Kitzes, 1983 ).

Mg deficiency can perturb calcium homeostasis and

result in a fall in both serum PTH and 1,25(OH) 2 D as

discussed earlier ( Rude et al., 1978 ; Fatemi et al., 1991 ).

Because 1,25(OH) 2 D stimulates osteoblast activity

( Azria, 1989 ) and the synthesis of osteocalcin and pro-

collagen ( Francheschi et al., 1988 ), decreased formation

of 1,25(OH) 2 D may be a major cause of decreased bone

formation, such as that observed in experimental Mg defi-

ciency ( Heroux et al., 1975 ; Jones et al., 1980 ; Kenney

et al., 1994 ). A decrease in bone-specific binding sites

for 1,25(OH) 2 -D has also been described, which could

account for vitamin D resistance ( Risco and Traba, 2004 ).

Similarly, PTH has been demonstrated to be trophic for

bone ( Marcus, 1994 ) and therefore impaired PTH secretion

or PTH skeletal resistance may result in osteoporosis.

Because insulin promotes amino acid incorporation

into bone ( Hahn et al., 1971 ), stimulates collagen produc-

tion ( Wettenhall et al., 1969 ), and increases nucleotide

synthesis by osteoblasts ( Peck and Messinger, 1970 ), insu-

lin deficiency or resistance may alter osteoblast function in

diabetes. However, insulin also causes an increase in intra-

cellular Mg, and because Mg has been shown to be trophic

for the osteoblast ( Liu et al., 1988 ), insulin deficiency may

result in intracellular Mg depletion and impaired osteoblast

activity. Serum IGF-1 levels have also been observed to

be low in the Mg-deficient rat, which could affect skeletal

growth ( Dorup et al., 1991 ).

Although the explanation just given may explain low

bone formation, it does not explain the observation of an

increase in osteoclast bone resorption. Each year about

25% of trabecular bone is resorbed and replaced in human

adults, whereas only 3% of cortical bone undergoes remod-

eling ( Papanicolou et al ., 1998 ). This suggests that the

rate of locally controlled bone remodeling is important

in the development of osteoporosis. Mg has been shown

to inhibit the N-methyl-D-aspartate (NMDA) recep-

tor ( McIntosh, 1993 ). Activation of the NMDA receptor

induces the release of neurotransmitters, such as substance

P ( McIntosh, 1993 ). Reduction of extracellular Mg low-

ers the threshold level of excitatory amino acids (i.e., glu-

tamate) necessary to activate this receptor. In one study,

dietary Mg deficiency produced raised serum levels of neu-

ropeptides such as substance P and calcitonin gene related

protein in rodents (Weglicki et al., 1996a) . This neuro-

genic response is followed by release of proinflammatory

4 weeks

Nu

mb

er

of

oste

ocla

sts

pe

rm

m t

rab

ecu

lar

bo

ne

su

rfa

ce

16 weeks

10

8

6

4

2

0

*

** # .01p

FIGURE 13 After 4 and 16 weeks of magnesium deficiency in the rat

(solid bars), the osteoclast number was elevated significantly compared to

controls (open bars) ( Rude et al., 1999 ) * p , 0.01.

10

20

30

40

C MgD

C MgD

C MgD

BV

/TV

(%

)

a

b

20

40

60

80

TT

h,

um

a

b

1

2

3

4

Tb

N

ab

FIGURE 14 Effect of a low Mg diet at 10% of nutrient requirement on

trabecular bone volume (BV/TB), mean trabecular width (TTh), and tra-

becular number (TbN) in rats fed Control (C) or low magnesium (MgD)

diets as determined by microcomputed tomography. Values are means 6

SD, n 5 7. Bars without a common letter differ (p , 0.01). ( Rude et al.,

2004 ).


cytokines, such as TNF α , IL-1 β , IL-6, by T lymphocytes

during the first week of dietary Mg depletion ( Weglicki

et al., 1996a ; Weglicki et al ., 1996b ; Kramer et al ., 1997 ).

Many of the actions of substance P are mediated through

the neurokinin 1 (NK-1) receptor. Elevated plasma cyto-

kines and the inflammatory cardiac lesions observed in

Mg-deficient rats have been shown to be prevented by

administration of a NK-1 receptor antagonist ( Weglicki

et al ., 1996a ; Weglicki et al., 1996b ; Kramer et al. , 1997 ).

Studies have demonstrated that there are nerve fibers con-

taining a number of neuropeptides including substance P

in bone ( Lerner, 2000 ). Substance P has also been shown

to increase the release of IL-1 β and IL-6 by bone marrow

cells ( Rameshwar et al ., 1994 ). These cytokines, which

are systemically released as well as locally produced in the

bone microenvironment, are known to stimulate the recruit-

ment and activity of osteoclasts and increase bone resorp-

tion ( Miyaura et al ., 1995 ; Nanes, 2003 ). Increased amount

of substance P, TNF α , and IL-1 β have been found in Mg-

depleted mice ( Rude et al ., 2003 ) and/or rats ( Rude et al .,

2004 ; Rude et al ., 2005 ; Rude et al ., 2006 ). These cytokines

could contribute to an increase in osteoclastic bone resorp-

tion and explain the uncoupling of bone formation and

bone resorption observed in the rat. Increased production

of these cytokines has been implicated in the development

of sex steroid deficiency or postmenopausal osteoporosis

( Pacifici, 1996 ; Manolagas et al ., 1995 ). Evidence also sug-

gests that inducible nitric oxide synthetase is stimulated by

these cytokines and may mediate localized bone destruction

associated with metabolic bone diseases ( Ralston, 1994 ;

Brandi et al., 1995 ). Mg-deficient rodents have increased

free radical formation, which may affect the cytokine cas-

cade and influence skeletal metabolism ( Kramer et al .,

1997 ). These cytokines could contribute to an increase in

osteoclastic bone resorption and explain the uncoupling

of bone formation and bone resorption observed in the rat

( Rude et al. , 1999 ; Rude et al ., 2004 ; Rude et al ., 2005 ;

Rude et al. , 2006 ). The final pathway of osteoclastogenesis

has been proposed to involve three constituents of a cyto-

kine system: receptor activator of nuclear factor kB ligand

(RANKL); its receptor, receptor activator of nuclear factor

kB (RANK); and its soluble decoy receptor, osteoprotegerin

(OPG) ( Hofbauer and Heufelder, 2000 ; Manolagas, 2000 ).

RANKL is a membrane-bound, cytokine-like molecule that

is expressed in preosteoblastic cells. It stimulates the dif-

ferentiation, survival, and fusion of osteoclastic precursor

cells to activate mature RANK expressed in hematopoietic

osteoclast progenitors, and serves as an essential factor for

osteoclastic differentiation and activation. RANKL binds

to RANK with high affinity and this interaction is essen-

tial for osteoclastogenesis. OPG is expressed in a variety of

cell types, however, in bone it is mainly produced by cells

of osteoblastic lineage. OPG has very potent inhibitory

effects on osteoclast formation. It acts like a decoy receptor

and blocks the RANKL/RANK interaction ( Hofbauer and

Heufelder, 2000 ). The relative presence of RANKL and

OPG therefore dictates osteoclast bone resorption activity.

Osteoclasts can be formed or activated in a RANKL and/

or a RANKL-independent mechanism by TNF α ( Hofbauer

and Heufelder, 2001 ; Horowitz et al ., 2001 ; Nanes, 2003 ).

The immunohistochemical presence of these two cytokines

in Mg-deficient vs. control animals was examined and a

decrease in OPG and an increase in RANKL were observed

(Rude et al., 2005) . Whether or not these mechanisms for

Mg-induced bone loss are valid for suboptimal chronic

dietary Mg deficit in human osteoporosis is unknown.

Magnesium and Mineral Formation

Mg may also independently influence bone mineral forma-

tion. In in vitro studies, Mg has been shown to bind to the

surface of hydroxyapatite crystals and to retard the nucle-

ation and growth of hydroxyapatite and its precrystalline

intermediate, amorphorous calcium phosphate ( Blumenthal

et al., 1977 ; Bigi et al., 1992 ; Sojka and Weaver, 1995 ).

Mg has also been demonstrated to compete with calcium

for the same absorption site on hydroxyapatite ( Aoba et al.,

1992 ). Therefore, surface-limited Mg may play a role in

modulating crystal growth in the mineralization process.

In vivo studies have demonstrated that as the Mg con-

tent of bone decreases, the hydroxyapatite crystal size

increases, whereas high Mg content results in smaller

crystals. Rats fed excess Mg have smaller mineral crys-

tal in their bone than control pair-fed animals ( Burnell

et al., 1986 ). In contrast, Mg-deficient rats have a signifi-

cant increase in hydroxyapatite crystal size ( Boskey et al.,

1992 ). Clinical studies are also consistent with this effect

of Mg on crystal formation. A crystallinity index deter-

mined by infrared spectrophotometry has shown larger and

more perfect bone mineral crystals along with decreased

bone Mg in bone samples obtained from patients with dia-

betes mellitus, postmenopausal osteoporosis, and alcoholic

osteoporosis ( Blumenthal et al., 1977 ; Cohen and Kitzes,

1981 ; Cohen et al., 1983 ; Sojka and Weaver, 1995 ).

These conditions are known to have a high incidence of

Mg depletion. In contrast, uremic patients, characterized

by high serum Mg levels, have smaller, less perfect crys-

tals and high bone Mg ( Blumenthal et al., 1977 ; Cohen

and Kitzes, 1981 ; Cohen et al., 1983 ; Sojka and Weaver,

1995 ). An inverse correlation was found to exist between

bone Mg and crystallinity index. The effect of these find-

ings on crystallization in terms of bone strength and bone

metabolism has yet to be elucidated.

Mg may also have another indirect effect on crystalliza-

tion by influencing both osteocalcin formation and osteo-

calcin binding to hydroxyapatite. Osteocalcin has been

shown to inhibit the conversion of brushite to hydroxyapa-

tite and the nucleation of mineral formation ( Wians et al.,

1990 ). Therefore, the decrease in osteocalcin production, as


suggested by decreased serum and bone osteocalcin, in Mg

depletion may influence mineralization. However, Mg has

also been demonstrated to inhibit the binding of osteocal-

cin to hydroxyapatite by reducing the number of available

hydroxyapatite-binding sites for osteocalcin ( Wians et al.,

1983 ). Maximal inhibition occurred at 1.5 m M Mg, which

is within the physiologically relevant concentration range.

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Chapter 1

INTRODUCTION

Complex, free-living terrestrial organisms, such as humans,

maintain their level of extracellular ionized calcium

( Cao21 ) within a narrow range of about 1.1 to 1.3 m M

( Bringhurst et al., 1998 ; Brown, 1991 ). This near con-

stancy of Cao21 ensures that Ca 2 1 ions are available for

their extracellular roles, including serving as a cofactor for

clotting factors, adhesion molecules, and other proteins and

controlling neuronal excitability ( Brown, 1991 ). Moreover,

calcium and phosphate salts form the mineral phase of

bone, which affords a rigid framework that protects vital

bodily structures and permits locomotion and other move-

ments. The skeleton also provides a nearly inexhaustible

reservoir of these ions when their availability in the diet is

insufficient for the body’s needs ( Bringhurst et al ., 1998 ).

In contrast to Cao21 , the cytosolic-free calcium con-

centration ( Ca i21 ) has a basal level — about 100 n M — that

is nearly 10,000-fold lower ( Berridge et al ., 2003 ). Ca i21 ,

however, can increase 10-fold or more when cells are stimu-

lated by extracellular signals acting on their respective cell

surface receptors as a result of influx of Ca 2 1 and/or its

release from intracellular stores ( Berridge et al ., 2003 ).

Ca i21 plays central roles in regulating cellular processes as

varied as muscular contraction, cellular motility, differen-

tiation and proliferation, hormonal secretion, and apoptosis

( Berridge et al ., 2003 ). Because all intracellular Cao21 ulti-

mately originates from that present in the extracellular flu-

ids (ECF), maintaining near constancy of Cao21 also ensures

that this ion is available for its myriad intracellular roles.

The level of Cao21 is maintained by a homeostatic mech-

anism in mammals that comprises the parathyroid glands,

calcitonin (CT)-secreting C cells, kidney, bone, and intes-

tine ( Bringhurst et al ., 1998 ; Brown, 1991 ). Key elements

of this system are cells that are capable of sensing small

Chapter 26

deviations in Cao21 from its usual level and responding in

ways that normalize it ( Brown, 1991 ). Calcium ions have

long been known to traverse the plasma membrane through

various types of ion channels and other transport mecha-

nisms ( Berridge et al ., 2003 ); however, the actual mecha-

nism by which Cao21 was “ sensed ” remained an enigma for

many years. This chapter provides an update on our pres-

ent understanding of the process of Cao21 sensing, which

has increased greatly over the past 10 to 15 years, espe-

cially as it relates to the mechanisms that maintain Cao21

homeostasis.

It is becoming increasingly clear that Cao21 serves as a

versatile extracellular first messenger — in many instances

acting via the Ca 2 1 -sensing receptor (CaR) — that controls

numerous physiological processes beyond those govern-

ing Cao21 homeostasis (for review, see Tfelt-Hansen and

Brown, 2005 ). Although it is beyond the scope of this

chapter to review the “ nonhomeostatic ” roles of the CaR

in detail, an emerging body of evidence supports the con-

cept that the receptor participates in important interactions

between the system regulating Cao21 metabolism and other

homeostatic systems (i.e., that controlling water metabo-

lism). These interactions may be crucial for the successful

adaptation of complex life forms to the terrestrial environ-

ment. This chapter will likewise address these recently

emerging homeostatic relationships.

CLONING OF THE CaR

Just 15 years ago, the concept that there was a specific Cao

21 -sensing “ receptor ” was only supported by indi-

rect evidence derived from studies of a limited num-

ber of Cao21 -sensing cells, particularly parathyroid cells

( Brown, 1991 ; Juhlin et al ., 1987 ; Nemeth and Scarpa,

1987 ; Shoback et al ., 1988 ). It was necessary, therefore,

in devising a strategy for cloning the putative receptor

to employ an approach that detected its Cao21 -sensing

Biology of the Extracellular Ca 2 1 -Sensing Receptor

Edward M. Brown *

Endocrine-Hypertension Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

*Corresponding author: Edward M. Brown, EBRC 223A, 221 Longwood

Ave, Boston, MA0211, E-mail: [email protected]


activity using a bioassay — namely, expression cloning in

Xenopus laevis oocytes. Racke et al . (1993) and Shoback

and co-workers ( Chen et al ., 1994 ) both demonstrated

that X. laevis oocytes became responsive to Cao21 -sensing

receptor agonists after they were injected with messenger

RNA (mRNA) extracted from bovine parathyroid glands.

Brown et al. (1993) were then able to utilize this strategy

to isolate a full-length, functional clone of the Cao21 -

sensing receptor. The use of conventional, hybridization-

based approaches subsequently permitted the cloning of

cDNAs coding for CaRs from human parathyroid ( Garrett

et al ., 1995b ) and kidney ( Aida et al., 1995b ), rat kid-

ney ( Riccardi et al ., 1995 ), brain (namely striatum; Ruat

et al ., 1995 ), and C cell ( Garrett et al., 1995c ); rabbit kid-

ney ( Butters et al ., 1997 ); and chicken parathyroid ( Diaz

et al ., 1997 ; reviewed in Brown et al ., 1999 ). All exhibit

very similar predicted structures and represent tissue and

species homologues of the same ancestral gene.

PREDICTED STRUCTURE OF THE CAR AND

ITS RELATIONSHIPS TO OTHER

G PROTEIN-COUPLED RECEPTORS

The topology of the CaR protein predicted from its

nucleotide sequence is illustrated in Figure 1 . It has three

principal structural domains, which include (a) a large,

~600-amino-acid extracellular amino-terminal domain

(ECD), (b) a “ serpentine ” seven membrane-spanning motif

that is characteristic of the superfamily of G protein-coupled

receptors (GPCRs), and (c) a sizable carboxyl-terminal (C-)

tail of about 200 amino acids. Several GPCRs have been

identified that share striking topological similarities with

the CaR, particularly in their respective, large ECDs, as

well as a modest (20 2 30%) overall degree of amino acid

identity. These structurally related GPCRs are designated

family C receptors ( Kolakowski, 1994 ; Brauner-Osborne

et al ., 2007 ). The mammalian family C receptors include

SP

NH2

HS

P

P

P

P P

PCysteine N-glycosylation

613

635

670

650

683

700

745

725

770

792

828

807

838

862

PKC siteConserved

Acidic

HOOC

FIGURE 1 Predicted structure of the CaR (see text for additional details). SP, signal peptide; HS, hydrophobic segment. Reproduced with permission

from Brown et al . (1993) .

535Chapter | 26 Biology of the Extracellular Ca2+-Sensing Receptor

eight metabotropic glutamate receptors (mGluRs), two

GABA B receptors, the CaR, 3 taste receptors (T1R1-T1R3),

the V2R pheromone receptors, and a promiscuous amino

acid-sensing receptor, GPRC6A ( Christiansen et al ., 2007 ),

that may also serve as a second Cao21 -sensing receptor

(see later discussion; Pi et al ., 2005 ).

mGluRs are activated by glutamate, the major excit-

atory neurotransmitter in the central nervous system (CNS;

Nakanishi, 1994 ). The GABA B receptors are GPCRs that

recognize γ -aminobutyric acid (GABA), the principal

inhibitory neurotransmitter of the CNS ( Kaupmann et al .,

1997 ; Ng et al ., 1999 ). The putative pheromone recep-

tors reside solely in neurons of the vomeronasal organ in

rodents (VNO; Matsunami and Buck, 1997 ), which con-

trols instinctual behavior, via input from environmental

pheromones. The taste receptors enable us to taste sweet

substances (and artificial sweeteners) as well as so-called

“ umami ” (i.e., the taste of monosodium glutamate; Hoon

et al ., 1999 ; Xu et al ., 2004 ).

Therefore, all of the family C GPCRs share the prop-

erty of having small molecules as their ligands that pro-

vide environmental cues (i.e., pheromones) or serve as

extracellular messengers within the CNS (e.g., glutamate

or GABA) or in bodily fluids more generally (i.e., Cao21 ,

amino acids). As detailed later, ligands of the CaR and the

other family C GPCRs are thought to bind principally to

their respective ECDs. In contrast, most other GPCRs bind-

ing small ligands, such as epinephrine or dopamine, have

binding sites within their TMDs and / or extracellular loops

(ECLs). The ligand-binding capacity of the family C ECDs

probably has its origin in a family of extracellular binding

proteins in bacteria (Felder et al ., 1999) , the so-called peri-

plasmic binding proteins (PBPs). These serve as receptors

for a wide variety of small ligands present in the environ-

ment, including ions (including Mgo21 , but apparently not

Cao21 ), amino acids, and other nutrients ( Tam and Saier,

1993 ). PBPs promote bacterial chemotaxis toward these

environmental nutrients and other substances and facilitate

their cellular uptake by activating specific transport sys-

tems in the cell membrane ( Tam and Saier, 1993 ).

The family C GPCRs, therefore, can be thought of as

representing fusion proteins, which comprise an extracel-

lular ligand-binding motif (the ECD) linked to a signal-

transducing motif (the seven transmembrane domains) that

couples the sensing of extracellular signals to intracellu-

lar signaling systems (i.e., G proteins and their associated

second messenger pathways). It is of interest that some of

the biological functions regulated by the CaR are the same

as those controlled by the PBPs, namely chemotaxis (e.g.,

of monocytes toward elevated levels of Cao21 ; Sugimoto

et al ., 1993 ) and cellular transport (i.e., of Cao21 by

CaR-regulated, Ca21-permeable channels; Chang et al .,

1995 ). Moreover, as described later, the CaR binds not

only Cao21 but also additional ligands, including amino

acids ( Conigrave et al ., 2000 ), which further supports its

evolutionary and functional relationships to the other fam-

ily C GPCRs and, ultimately, to PBPs.

BIOCHEMICAL PROPERTIES OF THE CaR

Studies using chimeric receptors comprising the ECD of

the CaR coupled to the TMDs and C tail of the mGluRs

(and vice versa) have demonstrated the existence of key

binding sites for Cao21 within the CaR’s ECD ( Brauner-

Osborne et al., 1999 ; Silve et al ., 2005 ), although even a

receptor lacking the entire ECD still has the capacity to

respond to polyvalent cations (Hu and Spiegel, 2003) . Studies

have indicated that specific residues within the ECD (e.g.,

Ser147 and Ser170) may be involved, directly or indirectly,

in the binding of Cao21 ( Silve et al., 2005 ; Huang et al .,

2007b ). Given the apparent “ positive cooperativity ” of the

CaR and the resultant steep slope of the curve describing

its activation by various polycationic agonists (e.g., Cao21

and Mgo21 ; Brown, 1991 ), however, it is likely that the

CaR binds several calcium ions. This positive cooperativ-

ity is essential to ensure that the CaR responds over the

narrow range of Cao21 regulating, for instance, PTH secre-

tion. This cooperativity could result, at least in part, from

the presence of Cao21 -binding sites on both of the individ-

ual ECDs within the dimeric CaR (see later discussion) as

well as the existence of more than one binding site within

each receptor monomer. Further work is needed, therefore,

in defining more precisely the identity of the CaR’s Cao21 -

binding site(s). Eventual solution of the three-dimensional

structure of the receptor’s ECD by x-ray crystallography

will no doubt illuminate how the CaR binds Cao21 and its

other agonists and modulators.

As noted earlier, the CaR resides on the cell surface

primarily in the form of a dimer ( Bai et al., 1998a ; Ward

et al ., 1998 ). CaR monomers within the dimeric recep-

tor are linked by disulfide bonds within their ECDs that

involve the cysteines at amino acid positions 129 and 131

( Ray et al ., 1999 ). Moreover, functional interactions can

occur between the monomeric subunits of the dimeric

CaR because two individually inactive CaRs that harbor

inactivating mutations in different functional domains

(e.g., the ECD and C tail) can reconstitute substantial bio-

logical activity when they heterodimerize after cotrans-

fection in human embryonic kidney (HEK293) cells ( Bai

et al ., 1999 ). Therefore, even though the individual CaRs

lack biological activity, they “ complement ” one another’s

defects through a mechanism that must involve intermo-

lecular interactions to form a partially active heterodimer.

In addition to forming homodimers with itself, the CaR

can also heterodimerize with the mGluRs (Gama et al .,

2001) as well as the GABA B receptors ( Cheng et al ., 2006 ).

In the latter case, heterodimerization changes the cell sur-

face expression as well as the functional properties of the

CaR ( Cheng et al ., 2006 ).


The ECD of the CaR on the cell surface is N-

glycosylated extensively with complex carbohydrates ( Bai

et al ., 1996 ). Eight of the predicted N-glycosylation sites in

the ECD of the human CaR are glycosylated ( Ray et al .,

1998 ). Disrupting four or five of these sites decreases the

cell surface expression of the receptor by 50% to 90%.

Therefore, glycosylation of at least three sites is required

for robust cell surface expression, although glycosylation

per se does not appear to be critical for the capacity of the

CaR to activate its intracellular signaling pathways ( Ray et al .,

1998 ).

Within its intracellular loops and C tail, the human CaR

harbors five predicted protein kinase C (PKC) and two pre-

dicted protein kinase A (PKA) phosphorylation sites ( Bai

et al., 1998b ; Garrett et al ., 1995b ). Activation of PKC

diminishes CaR-mediated stimulation of phospholipase

C (PLC), and studies utilizing site-directed mutagenesis

have demonstrated that phosphorylation of a single, key

PKC site in the C tail of the CaR at Thr888 can account for

most of the inhibitory effect of PKC on the function of the

receptor ( Bai et al ., 1998b ; Davies et al ., 2007) . Therefore,

PKC-induced phosphorylation of the C tail may afford a

means of conferring negative feedback regulation on the

coupling of the receptor to PLC. That is, PLC-elicited acti-

vation of protein kinase C — through the ensuing phosphor-

ylation of the CaR at Thr888 — limits further activation of

this pathway. The functional significance of the PKA sites

is not fully elucidated, although one study found that PKA

synergized with PKC in inhibiting CaR-mediated activa-

tion of PLC ( Bosel et al ., 2003 ).

BINDING PARTNERS OF THE CaR AND

THEIR BIOLOGICAL ROLES

A number of molecular binding partners of the CaR have

been identified that provide insights into the molecular

details of the receptor’s trafficking to the cell surface, its

signaling and other biological functions, and its desensi-

tization and degradation. During its initial biosynthesis, the

capacity of the CaR to successfully reach the cell surface

depends, at least in some cells, on its capacity to bind to

receptor activity-modifying proteins (RAMPs; Bouschet

et al ., 2005 ). The CaR in parathyroid and likely in other

cells resides within caveolae, flask-like invaginations of

the plasma membrane ( Kifor et al ., 1998 ), where it binds

directly or indirectly to caveolin-1, a key structural pro-

tein within caveolae that binds to multiple other pro-

teins, including signaling molecules such as G proteins

( Williams and Lisanti, 2004 ). The C tail of the cell surface

CaR interacts with filamin-A, an actin-binding protein that,

like caveolin-1, serves a scaffolding function by binding

multiple classes of proteins (Hjalm et al ., 2001) ; ( Awata

et al ., 2001 ). The CaR’s capacity to activate MAPKs, such as

ERK1 / 2 (see next section) depends, in part, on its physical

interaction with filamin-A (and likely the latter’s capacity

to bind various MAPK components; Hjalm et al ., 2001;

( Awata et al ., 2001 ). The CaR’s capacity to activate the

low-molecular-weight G protein, Rho, via the Rho guanine

nucleotide exchange factor, RhoGEF, also depends on the

receptor’s binding to filamin-A ( Pi et al ., 2002 ). The CaR’s

C tail has recently been shown to bind to two potassium

channels, Kir4.1 and 4.2, an interaction that inhibits the activ-

ities of these channels ( Huang et al ., 2007a ). The CaR is

noteworthy for the limited extent to which it is desensitized

on repeated exposure to Cao21 and other polycationic ago-

nists , e.g., in parathyroid cells, which depends, in part, on

its tethering to the actin-based cytoskeleton by filamin-A

( Zhang and Breitwieser, 2005 ). However, it does desensi-

tize in heterologous systems following its phosphorylation

by (1) PKC, which reduces the CaR’s capacity to activate

PLC or (2) G protein-coupled receptor kinases (GRK) and

subsequent binding of the phosphorylated receptor to the

adapter protein, β -arrestin ( Lorenz et al ., 2007 ). The lat-

ter decreases its capacity to activate G proteins. Although

there is only limited information on the fate of the desensi-

tized receptor, a portion of it can undergo proteasomal deg-

radation after binding to the ubiquitin ligase, dorfin, and

subsequent ubiquitination ( Huang et al ., 2006 ).

INTRACELLULAR SIGNALING BY THE CaR

Activation of the CaR by its agonists stimulates the activi-

ties of phospholipases C, A 2 (PLA 2 ), and D (PLD) in

bovine parathyroid cells and in HEK293 cells stably trans-

fected with the human CaR ( Kifor et al ., 1997 ). In most

cells, CaR-evoked activation of PLC involves the partici-

pation of the pertussis toxin-insensitive G protein(s), G q/11

( Brown and MacLeod, 2001 ), although in some it can take

place through a pertussis toxin-sensitive pathway, most

likely via one or more isoforms of the G i subfamily of G

proteins ( Emanuel et al ., 1996 ). In bovine parathyroid cells

and CaR-transfected HEK293 cells, CaR-mediated stimu-

lation of PLA 2 and PLD occurs through PKC-dependent

mechanisms, presumably via receptor-dependent activa-

tion of PLC ( Kifor et al ., 1997 ), although some studies

have shown that CaR-mediated activation of PLD involves

G 12/13 ( Huang et al ., 2004 ).

The high Cao21 -induced, transient increase in Ca i

21

in bovine parathyroid cells and CaR-transfected HEK293

cells likely results from activation of PLC ( Kifor et al .,

1997 ) and resultant IP 3 -mediated release of intracellu-

lar Ca 2 1 stores ( Bai et al ., 1996 ). High Cao21 also elic-

its sustained elevations in Ca i21 in both CaR-transfected

HEK293 cells ( Bai et al ., 1996 ) and bovine parathyroid

cells ( Brown, 1991 ), acting through an incompletely

defined influx pathway(s) for Ca 2 1 . Via the patch-clamp

technique, we have shown that the CaR activates a

Cao21 -permeable, nonselective cation channel (NCC) in


CaR-transfected HEK cells ( Ye et al ., 1996 ). An NCC with

similar properties is present in bovine parathyroid cells and

is likewise activated by high Ca 2 1 — an effect presumably

mediated by the CaR ( Chang et al ., 1995 ). This latter NCC

may participate in the high Cao21 -induced, sustained ele-

vation in Ca i21 in parathyroid cells ( Brown et al ., 1990 ).

Activation of the CaR by Cao21 initiates oscillations in

Ca i21 in several cell types ( Breitwieser and Gama, 2001 ;

Young and Rozengurt, 2002 ; De Luisi and Hofer, 2003 ),

through a mechanism involving activation of PLC (Rey

et al ., 2005) and the Ca 2 1 -permeable channel, TRPC1 ,

and this process can be modulated by PKC ( Young et al .,

2002 ). Amino acids stimulate a different pattern of oscil-

lations ( Young and Rozengurt, 2002 ), indicating that the

nature of the CaR agonist can affect the signals generated

by the receptor.

High Cao21 reduces agonist-stimulated cAMP accumu-

lation in bovine parathyroid cells markedly ( Chen et al .,

1989 ; Gerbino et al ., 2005 ; Corbetta et al ., 2000 ), an action

that is thought to involve inhibition of adenylate cyclase

by one or more isoforms of G i , as it is pertussis toxin-

sensitive ( Chen et al ., 1989 ). Other cells, however, can

exhibit high Cao21 -elicited diminution of cAMP accumu-

lation that involves indirect pathways, including inhibition

of a Ca i21 -inhibited isoform of adenylate cyclase due to

the associated rise in Ca i21 ( de Jesus Ferreira et al ., 1998 )

or by increasing the degradation of cAMP ( Geibel et al .,

2006 ). The CaR also activates mitogen-activated protein

kinases (MAPK) in several types of cells, including rat-

1 fibroblasts ( McNeil et al ., 1998b ), ovarian surface cells

( McNeil et al ., 1998a ), and CaR-transfected but not non-

transfected HEK293 cells ( Kifor et al ., 2001 ). As is the

case with other GPCRs, the CaR stimulates the activity

of MAPK (e.g. extracellar signal regulated kinases 1and 2

and p38 MAPK) through both PKC- and tyrosine kinase-

dependent pathways. The latter involves, in part, c-Src-like

cytoplasmic tyrosine kinases. The PKC-dependent activa-

tion of MAPK is presumably downstream of Gq-mediated

activation of PLC, whereas that involving tyrosine kinases

may utilize the Gi-dependent pathway involving β γ sub-

units released as a consequence of the activation of this G

protein ( Kifor et al ., 2001 ; McNeil et al ., 1998b ).

THE CaR GENE AND ITS REGULATION

Relatively little is presently known regarding the structure

of the CaR gene, especially its upstream regulatory regions

and the factors controlling its expression. The human CaR

gene is on the long arm of chromosome 3, as demon-

strated by linkage analysis ( Chou et al., 1992 ), and in band

3q13.3 2 21, as documented by fluorescent in situ hybrid-

ization ( Janicic et al., 1995 ). The rat and mouse CaR genes

reside on chromosomes 11 and 16, respectively ( Janicic

et al., 1995 ). The CaR gene has seven exons: The first

includes the upstream untranslated region, the next five

encode various regions of the ECD, and the last encodes

the rest of the CaR from its first TMD to the C terminus

( Pearce et al., 1995 ). Characterization of the upstream reg-

ulatory regions of the gene will be of great interest because

expression of the CaR can change in a various physiologi-

cally relevant circumstances — some of which are delin-

eated hereafter.

Several factors increase CaR expression. Both high

Cao21 and 1,25(OH) 2 D 3 upregulate expression of the gene

in certain cell types. High Cao21 increases expression of

the CaR in ACTH-secreting, pituitary-derived AtT-20 cells

( Emanuel et al., 1996 ) as well as in avian (Yarden et al.,

2000) and human parathyroid glands (Mizobuchi et al.,

2004) , whereas administration of 1,25(OH) 2 D 3 elevates

expression of the CaR in vivo in kidney and parathyroid

gland of the rat in some ( Brown et al., 1996 ; Canaff and

Hendy, 2002 ) but not all studies ( Rogers et al., 1995 ).

Interleukin-1 β increases the level of CaR mRNA modestly

in bovine parathyroid gland fragments ( Nielsen et al., 1997 )

as well as in rat parathyroid and kidney ( Canaff and Hendy,

2005 ), which may contribute to the associated reduction in

PTH secretion that was observed in the former study. In rat

kidney, substantial upregulation of the CaR takes place dur-

ing the perinatal and immediate postnatal period; the resul-

tant higher level of expression of the receptor then persists

throughout adulthood ( Chattopadhyay et al., 1996 ). There

is likewise a developmental increase in expression of the

CaR in rat brain. In contrast to that occurring in the kid-

ney, however, the rise in the expression of the CaR in the

brain occurs about a week postnatally ( Chattopadhyay

et al., 1997 ). Moreover, the increase in CaR expression

in the brain is only transient — it decreases severalfold

approximately 2 weeks later and reaches a lower level that

remains stable thereafter ( Chattopadhyay et al., 1997 ). The

biological importance of these developmental changes in

the expression of the receptor is currently unknown. The

CaR is overexpressed in some cancers (Li et al., 2005) , and

it has been suggested to be a marker of breast cancer cells

that have a propensity for metastasis to bone ( Mihai et al.,

2006 ) to act as a factor contributing to lytic bone disease in

animal models of prostate cancer ( Liao et al., 2006 ).

In contrast, several instances have been defined in which

CaR expression decreases. Calf parathyroid cells exhibit a

rapid, marked (80 2 85%) reduction in expression of the

receptor after they are placed in culture ( Brown et al., 1995 ;

Mithal et al., 1995 ), which is likely to be a major factor

that contributes to the associated reduction in high Cao21 -

evoked inhibition of PTH secretion. The expression of the

CaR in rat kidney decreases in a model of chronic renal

insufficiency induced by subtotal nephrectomy ( Mathias

et al., 1998 ). This latter reduction in CaR expression might

contribute to the hypocalciuria that occurs in human renal

insufficiency, as reduced renal CaR expression and / or

activity increases tubular reabsorption of Ca 2 1 in humans


with inactivating mutations of the receptor ( Brown, 1999 ).

Because 1,25(OH) 2 D 3 upregulates renal CaR expression

( Brown et al., 1996 ; Canaff and Hendy, 2005 ), the reduction

in CaR expression in the setting of impaired renal function

could be the result, in part, of the concomitant decrease in

circulating levels of 1,25(OH) 2 D 3 ( Bringhurst et al., 1998 ).

As in parathyroid cells, the CaR in several other cells types

is thought to serve as an inhibitor of cell proliferation, e.g.,

in cells from the colonic crypts ( Kallay et al., 2003 ) and

in keratinocytes ( Tu et al., 2004 ). Therefore, the reduced

expression in some colon cancers may contribute to tumori-

genesis ( Kallay et al., 2003 ; Bhagavathula et al ., 2005 ).

ROLES OF THE CaR IN TISSUES

MAINTAINING Cao21 HOMEOSTASIS

Parathyroid

The parathyroid glands of several species express high lev-

els of CaR mRNA and protein, including those of humans

( Kifor et al ., 1996 ), rats ( Autry et al ., 1997 ), mice ( Ho et al.,

1995 ), rabbits ( Butters et al., 1997 ), and chickens ( Diaz

et al., 1997 ). Indeed, the level of CaR expression in the para-

thyroid chief cells is one of the highest, if not the highest, in

the cells and tissues examined to date. Abundant evidence

supports the importance of the CaR as the key mediator of

the inhibitory action of elevated Cao21 on PTH secretion. As

noted earlier, the reduced CaR expression in bovine parathy-

roid cells maintained in culture is associated with a progres-

sive loss of high Cao21 -induced inhibition of PTH secretion

( Brown et al., 1995 ; Mithal et al., 1995 ). In addition, individ-

uals with familial hypocalciuric hypercalcemia (FHH), who

are heterozygous for inactivating mutations of the CaR gene

( Brown, 1999 ), or mice that are heterozygous for targeted

disruption of the CaR gene ( Ho et al., 1995 ) exhibit mod-

est right shifts in their set points for Cao21 -regulated PTH

secretion (the level of high Cao21 half-maximally inhibiting

PTH release), indicative of “ Cao21 resistance ” Moreover,

humans and mice homozygous for loss of the normal CaR

( Brown, 1999 ; Ho et al., 1995 ) show much greater impair-

ment of high Cao21 -elicited suppression of PTH release,

showing that that the “ Cao21 resistance ” of the parathyroid is

related inversely to the number of normally functioning CaR

alleles. Therefore, the biochemical findings in mice in which

the CaR has been “ knocked out, ” as well as in humans with

naturally occurring inactivating mutations of the CaR, prove

the central role of the CaR in Cao21 -regulated PTH release.

Despite several decades of study, however, the principal

intracellular signaling mechanism(s) through which the CaR

inhibits PTH secretion remains enigmatic (for review, see

Diaz et al., 1998) . However, recent evidence indicates that

activation of the G q/11 pathway is essential, since mice with

knockout of both of these G proteins have severe hyperpara-

thyroidism mimicking that present in mice homozygous for

knockout of the CaR itself (Wettschureck et al., 2007). By

a series of poorly defined steps, the CaR eventually induces

polymerization of the actin-based cytoskeleton in a way that

may provide a physical impediment to the release of PTH-

containing secretory vesicles ( Quinn et al., 2007 ).

Another aspect of parathyroid function that is likely

to be CaR regulated is PTH gene expression. Garrett et al.

(1995a) showed in preliminary studies that the “ calcimi-

metic ” CaR activator NPS R-568, which activates the recep-

tor allosterically through a mechanism involving an increase

in the apparent affinity of the CaR for Cao21 ( Nemeth et al.,

1998b ), decreases the level of PTH mRNA in bovine para-

thyroid cells. More recent studies ( Levi et al., 2006 ) have

documented that the same calcimimetic reduces the elevated

level of preproPTH mRNA in rats with experimentally

induced secondary hyperparathyroidism owing to subtotal

nephrectomy. This effect is post-transcriptional and involves

modulation of the binding of the transacting factor, AUF1, to

preproPTH mRNA. Finally, the CaR, directly or indirectly,

tonically inhibits the proliferation of parathyroid cells,

because persons homozygous for inactivating mutations of

the CaR ( Brown, 1999 ) or mice homozygous for “ knockout ”

of the CaR gene ( Ho et al., 1995 ) exhibit marked parathy-

roid cellular hyperplasia. Treatment of rats with experimen-

tally induced renal impairment with calcimimetics prevents

the parathyroid hyperplasia that would otherwise be antici-

pated to occur in this setting ( Wada et al., 1998 ; Colloton

et al., 2005 ), providing further evidence that activation of the

CaR suppresses parathyroid cellular proliferation.

C Cells

Unlike the effect of Cao21 on PTH release, elevating Cao

21

stimulates CT secretion — a response that conforms to the

classical, positive relationship between Cao21 and activa-

tion of exocytosis in most other hormone-secreting cells

( Bringhurst et al., 1998 ; Brown, 1991 ). This observation

was one of several pieces of indirect evidence that the

mechanism underlying Cao21 sensing in C cells might differ

in a fundamental way from that in parathyroid cells. More

recent data, however, have demonstrated that rat, human,

and rabbit C cells express the CaR ( Butters et al., 1997 ;

Freichel et al., 1996 ; Garrett et al., 1995c ). Moreover, clon-

ing of the CaR from a rat C cell tumor cell line showed it to

be identical to that expressed in rat kidney ( Garrett et al.,

1995c ). Available evidence, albeit limited, indicates that the

CaR mediates the stimulatory effect of high Cao21 on CT

secretion; namely, there is a shift to the right in the rela-

tionship between the serum calcium concentration and CT

levels in mice heterozygous for knockout of the CaR gene

(Fudge and Kovacs, 2004) . Tamir and co-workers have sug-

gested that the following sequence of steps mediates CaR-

stimulated CT secretion ( McGehee et al., 1997 ). High

Cao21 first stimulates phosphatidylcholine-specific PLC,


which generates diacylglycerol, thereby activating an NCC

through a PKC-dependent mechanism. The activated NCC

then enhances cellular uptake of Na 1 and Ca 2 1 , producing

cellular depolarization and consequent stimulation of volt-

age-dependent, principally L-type Ca 2 1 channels that elevate

Ca i21 and activate exocytosis of 5-hydroxytryptamine- and

CT-containing secretory granules.

Kidney

In the kidney of the adult rat, the CaR is expressed along

almost the entire nephron, with the highest levels of pro-

tein expression at the basolateral aspect of the epithelial

cells of the cortical thick ascending limb (CTAL; Riccardi

et al., 1998 ). The CTAL plays a key role in the hormone

(e.g., PTH-regulated reabsorption of divalent minerals ( De

Rouffignac and Quamme, 1994 ; Friedman and Gesek,

1995 )). The CaR is also present on the basolateral mem-

branes of the cells of the distal convoluted tubule (DCT),

where Ca 2 1 reabsorption — similar to that in the CTAL—is

stimulated by PTH. Further sites of renal CaR expression

include the base of the microvilli of the proximal tubular

brush border ( Riccardi et al., 1998 ), the basolateral sur-

face of the tubular cells of the medullary thick ascending

limb (MTAL; Riccardi et al., 1998 ), and the luminal side

of the epithelial cells of the inner medullary collecting

duct (IMCD; Sands et al., 1997 ). None of these latter three

nephron segments play major roles in renal Cao21 handling,

but the CaR expressed in them could conceivably modulate

the handling of other solutes and/or water. As will be dis-

cussed in more detail later, the CaR in the CTAL, in addi-

tion to modulating reabsorption of Ca 2 1 and Mg 2 1 , also

participates in controlling the renal handling of Na 1 , K 1 ,

and Cl – ( Hebert et al., 1997 ). Finally, the CaR expressed in

the IMCD likely mediates the well-recognized inhibitory

action of high Cao21 on vasopressin-evoked water reab-

sorption ( Sands et al., 1997, 1998 ), which is one cause of

the defective urinary-concentrating ability in some patients

with hypercalcemia ( Brown, 1999 ; Brown et al., 1999 ).

In the proximal tubule, activation of the CaR inhib-

its PTH-stimulated phosphaturia ( Ba et al., 2003 ) and

increases expression of the vitamin D receptor ( Maiti and

Beckman, 2007 ), which could conceivably contribute to the

direct, high calcium-induced lowering of 1,25(OH) 2 D 3 lev-

els in parathyroidectomized, PTH-infused rats (Weisinger

et al., 1989) . The localization of the CaR on the basolateral

membrane in the CTAL indicates that it might represent

the mediator of the previously demonstrated inhibitory

effect of high peritubular but not luminal levels of Cao21

on Ca 2 1 and Mg 2 1 reabsorption in perfused tubular seg-

ments from this portion of the nephron ( De Rouffignac and

Quamme, 1994 ). Figure 2 shows a schematic illustration

Ca21, Mg21

Ca21

Ca21

Na1

2CI2

CI2K1

K1

Stimulatestransport

P450

Lumen-positive voltage

Urinaryspace

Basolateralspace

Hormones

cAMP

H2O

1

3

4 2

PLA2

AA

1 2

FIGURE 2 Possible mechanisms by which the CaR modulates intracellular second-messenger pathways and ionic transport in the CTAL. Hormones

that elevate cAMP (i.e., PTH) stimulate paracellular Ca 2 1 and Mg2 1 reabsorption by increasing the activities of the Na 1 -K 1 -2Cl– cotransporter and an

apical K 1 channel and, therefore, increasing the magnitude of V t . The CaR, which like the PTH receptor is on the basolateral membrane, inhibits PTH-

stimulated adenylate cyclase and activates PLA2 (2). This latter action increases free arachidonic acid, which is metabolized by the P450 pathway to an

inhibitor of the apical K 1 channel (4) and, perhaps, the cotransporter (3). Actions of the CaR on both adenylate cyclase and PLA2 will reduce Vt and,

therefore, diminish paracellular divalent cation transport. Reproduced with permission from Brown and Hebert (1997a) .


of how the CaR may inhibit PTH-enhanced divalent cation

reabsorption in the CTAL. As indicated in detail in Figure 2 ,

the CaR acts in a “ Lasix-like ” fashion to diminish overall

activity of the Na 1 -K 1 -2Cl – cotransporter, which generates

the lumen-positive, transepithelial potential gradient that

normally drives passive paracellular reabsorption of about

50% of NaCl and most of the Ca 2 1 and Mg 2 1 in this part

of the nephron ( Hebert et al., 1997 ). Some studies, how-

ever, while demonstrating inhibition of calcium transport

in CTAL by elevating peritubular Cao21 , failed to show any

concomitant reduction in NaCl or magnesium transport or

in the transepithelial potential (DesFleurs et al., 1998) , or

else have documented inhibitory effects of peritubular cal-

cium on not only paracellular but also transepithelial Ca 2 1

transport ( Motoyama and Friedman, 2002 ). Further studies

are needed to resolve these differences. It is of interest that

individuals with FHH manifest a markedly reduced ability

to upregulate urinary excretion of Ca 2 1 despite their hyper-

calcemia — even when they have been rendered aparathy-

roid by total parathyroidectomy ( Attie et al ., 1983 ). Thus,

the PTH-independent, excessive reabsorption of Ca 2 1 in

FHH likely results, in part, from a decreased complement

of normally functioning CaRs in the CTAL. This defect

renders the tubule “ resistant ” to Cao21 and reduces the nor-

mal, high Cao21 -evoked hypercalciuria that occurs in this

nephron segment ( Brown, 1999 ). Thus, in normal persons,

hypercalcemia-evoked hypercalciuria likely has two dis-

tinct CaR-mediated components: (1) suppression of PTH

secretion and (2) direct inhibition of tubular reabsorption

of Ca 2 1 in the CTAL.

It is not presently known whether the CaR modulates

PTH-stimulated Ca 2 1 reabsorption in the DCT, which

could potentially occur via inhibition of PTH-stimulated

cAMP accumulation. In addition, extracellular Ca 2 1 has

been shown to modulate the expression of key, vitamin D—

inducible genes involved in transcellular Ca 2 1 trans-

port in this nephron segment, namely, the apical uptake

channel TRPV5, calbindin D and the basolateral cal-

cium pump, PMCA2b, and exchanger, NCX1 ( Thebault

et al., 2006 ). These changes take place both indirectly,

by high Ca 2 1 -elicited changes in PTH secretion and

pari passu 1,25(OH) 2 D 3 production, as well as by appar-

ently direct renal actions of Cao21 that are independent of

1,25(OH) 2 D 3 . The contribution of these actions of Cao21 to

the function of DCT in tubular reabsorption of Ca 2 1 under

normal circumstances is not currently known.

Intestine

The intestine is a key participant in maintaining Cao21

homeostasis by virtue of its capacity for regulated absorp-

tion of dietary Cao21 through the action of 1,25(OH) 2 D 3 ,

the most active naturally occurring metabolite of

vitamin D ( Bringhurst et al., 1998 ; Brown, 1991 ). The duo-

denum is the principal site for 1,25-dihydroxyvitamin D 3

[1,25(OH) 2 D 3 ]-stimulated intestinal Ca 2 1 absorption through

a transcellular pathway of active transport. The latter is

thought to involve initial Ca 2 1 entry through the recently

identified Ca 2 1 -permeable channel, TRPV6 (previously

known as CaT1 or ECaC2; Peng et al., 1999 ; Hoenderop

et al., 1999). As with the transcellular transport of Ca 2 1

in DCT, calcium ions subsequently diffuse across the

cell — a process facilitated by the vitamin D 2 dependent

Ca 2 1 -binding protein, calbindin D 9K — and are eventu-

ally extruded at the basolateral cell surface by the Ca 2 1 -

ATPase and, perhaps, the Na 1 -Ca 2 1 exchanger ( Thebault

et al., 2006 ). Jejunum and ileum absorb less Ca 2 1 than

duodenum (particularly when expressed as absorption

per unit surface area). They also secrete Ca 2 1 , which

may chelate fatty acids and bile acids, thereby producing

insoluble “ calcium soaps ” and mitigating possible dam-

aging effects of free fatty acids and bile salts on colonic

epithelial cells. The major function of the colon in fluid

and electrolyte metabolism is the absorption of water and

Na 1 . Nevertheless, it absorbs substantial amounts of Ca 2 1

in humans by both vitamin D-dependent and -independent

routes, especially in its proximal segments ( Favus, 1992 ),

where it expresses levels of TRPV6 similar to those in the

duodenum ( Peng et al., 1999 ).

The CaR is expressed in all segments of the intestine

in the rat. The highest levels of expression of the recep-

tor are on the basal surface of the small intestinal absorp-

tive cells, the epithelial cells of the crypts of both the

small intestine and colon, and in the enteric nervous sys-

tem ( Chattopadhyay et al., 1998 ). Does the CaR have any

role in systemic Cao21 homeostasis in these locations?

There are several direct actions of Cao21 on intestinal

function. Hypercalcemia inhibits dietary Ca 2 1 absorption

( Krishnamra et al., 1994 ). Recent studies have also shown

apparently direct actions of dietary and/or extracellular

Ca 2 1 on the expression of TRPV6, calbindin D 9K , and

PMCA2B in mice with knockout of the 1-hydroxylase

gene, which are of uncertain physiological significance at

present but document the capacity of the GI tract to sense

Cao21 ( Hoenderop et al., 2004 ). Moreover, direct measure-

ments of Cao21 within the interstitial fluid underneath small

intestinal absorptive epithelial cells has shown that Cao21

increases by nearly twofold if luminal levels of Cao21 are

elevated to 5 to 10 m M — similar to those achieved after

the intake of Ca 2 1 -containing foods ( Mupanomunda et al.,

1999 ). This level of Cao21 would be more than sufficient

to stimulate the CaR expressed on the basolateral surface

of small intestinal absorptive cells. The CaR expressed in

the enteric nervous system, which regulates the secretomo-

tor functions of the gastrointestinal tract, could potentially

mediate known effects of high and low Cao21 to reduce

and increase GI motility, respectively, in hyper- and hypo-

calcemic individuals ( Bringhurst et al., 1998 ). Such an

effect on gastrointestinal motility, however, even if it were

CaR-mediated, would not have any obvious relevance to


systemic Cao21

homeostasis, other than perhaps indirectly,

by modulating the time available for absorption of Ca 2 1

and other nutrients.

Bone and Cartilage

The levels of Cao21 achieved within the bony microenvi-

ronment probably vary substantially during the regulated

turnover of the skeleton via osteoclastic resorption of bone

followed by its restoration by bone-forming osteoblasts — a

process that totally replaces the human skeleton approxi-

mately every 10 years ( Bringhurst et al., 1998 ). In fact, Cao

21 directly beneath resorbing osteoclasts can be as high

as 8 to 40 m M (Silver et al., 1988). Moreover, Cao21 has a

variety of actions on bone cell functions in vitro that could

serve physiologically useful purposes, although it is not

yet known whether these same actions occur in vivo. For

example, high Cao21 stimulates parameters of osteoblastic

functions that could enhance their recruitment to sites of

future bone formation, including chemotaxis and prolifera-

tion, and promote their differentiation to osteoblasts with a

more mature phenotype ( Quarles, 1997 ; Yamaguchi et al.,

1999 ; Dvorak et al., 2004 ). Furthermore, elevated levels of

Cao21 suppress both the formation ( Kanatani et al., 1999 )

and the activity ( Zaidi et al., 1999 ) of osteoclasts in vitro.

Therefore, Cao21 has effects on cells of both the osteoblas-

tic and osteoclastic lineages and/or their precursors that

are homeostatically appropriate. Raising Cao21 would,

for example, produce net movement of Ca 2 1 into bone by

stimulating bone formation and inhibiting its resorption.

In addition, locally elevated levels of Cao21 produced by

osteoclasts at sites of active bone resorption could poten-

tially contribute to “ coupling ” bone resorption to the ensu-

ing replacement of the missing bone by osteoblasts, by

promoting proliferation and recruitment of preosteoblasts

within the vicinity to these sites and enhancing their dif-

ferentiation ( Quarles, 1997 ; Yamaguchi et al., 1999 ). As

detailed later, the molecular identity of the Cao21 -sensing

mechanism(s) in bone cells remain(s) controversial,

although the CaR has been found by several groups of

investigators to be present in at least some cells of both

osteoblast and osteoclast lineages and could, therefore,

potentially participate in this process.

Substantial indirect evidence amassed prior to and

around the time that the CaR was cloned suggested that the

Cao21 -sensing mechanism in osteoblasts and osteoclasts

differed in certain pharmacological and other properties

from those exhibited the CaR ( Quarles, 1997 ; Zaidi et al.,

1999 ). Moreover, some investigators have been unable to

detect expression of the CaR in osteoblast-like ( Pi et al.,

1999 ) and osteoclast-like cells ( Seuwen et al., 1999 ). More

recent studies, however, have provided strong support for

the presence of the CaR in a variety of cells originating

from the bone and bone marrow, although its physiological

and functional implications in these cells remain uncertain.

These CaR-expressing cells include hematopoietic stem cells

(Adams et al., 2006) , erythroid and platelet progenitors, cells

of the monocytic lineage ( Yamaguchi et al., 1998d ) [but not

granulocytes ( House et al., 1997 )], and cells of both the

osteoblast and osteoclast lineages when studied in situ in sec-

tions of bone ( Yamaguchi et al., 1999 ; Chang et al., 1999b ;

Dvorak et al., 2004 ). ST-2 stromal cells ( Yamaguchi et al.,

1998a ), a stromal cell line derived from the same mesen-

chymal stem cells giving rise to osteoblasts, express CaR

mRNA and protein, as do osteoblast-like cell lines (e.g.,

the Saos-2, MC-3T3-E1, UMR-106, and MG-63 cell lines;

Chang et al., 1999b ; Yamaguchi et al., 1998b, 1998c ).

Regarding cells of the osteoclast lineage, more than 80%

of human peripheral blood monocytes, which arise from

the same hematopoietic lineage that gives rise to osteo-

clast precursors, express abundant levels of CaR mRNA

and protein ( Yamaguchi et al., 1998d ). Preosteoclast-like

cells generated in vitro also show expression of the CaR

( Kanatani et al., 1999 ), and osteoclasts isolated from rab-

bit or mouse bone likewise express the receptor ( Kameda

et al., 1998 ; Mentaverri et al., 2006 ). In murine, rat, and

bovine bone sections, in contrast, only a minority of the

multinucleated osteoclasts expressed CaR mRNA and pro-

tein ( Chang et al., 1999b ). Additional studies are required

to clarify whether primarily osteoclast precursors, rather

than mature osteoclasts, express the CaR and whether

the CaR actually mediates some or even all of the known

actions of Cao21 on these cells. One study failed to detect

expression of the CaR in transformed osteoblast-like cells

derived from either wild-type mice or those with knock-

out of the CaR ( Pi et al., 2000 ). However, these cells still

showed some responses to Cao21 and Al 3 1 (e.g., mito-

genesis), suggesting the presence of another Cao21 sen-

sor, as this group has suggested in earlier studies ( Quarles

et al., 1997 ). This group has recently suggested that the

basic amino acid-sensing, family C receptor, GPRC6A,

represents the key Cao21 –sensing mechanism present in

osteoblasts ( Pi et al., 2005 ).

Although chondrocytes — the cells that form cartilage —

do not participate directly in systemic Cao21 homeostasis,

they play a key role in skeletal development and growth

by providing a cartilaginous model of the future skeleton

that is gradually replaced by actual bone. Furthermore, the

growth plate represents a site where chondrocytes play a

crucial role in the longitudinal growth that persists until the

skeleton is fully mature at the end of puberty. The avail-

ability of Ca 2 1 is important for ensuring proper growth

and differentiation of chondrocytes and resultant skeletal

growth in vivo ( Jacenko and Tuan, 1995 ; Reginato et al.,

1993 ). Moreover, changing the level of Cao21 in vivo mod-

ulates the differentiation and / or other properties of cells

of the cartilage lineage ( Bonen and Schmid, 1991 ; Wong

and Tuan, 1995 ). Chondrocytes arise from the same mes-

enchymal stem cell lineage that gives rise to osteoblasts,


smooth muscle cells, adipocytes, and fibroblasts ( Boyan

et al., 1999 ; Dennis et al., 1999 ). It is interesting, therefore,

that the rat cartilage cell line, RCJ3.1C5.18, showed readily

detectable levels of CaR mRNA and protein ( Chang et al.,

1999a ). In addition, some cartilage cells in sections of

intact bone express CaR mRNA and protein, including the

hypertrophic chondrocytes in the growth plate, which are

key participants in the growth of long bones ( Chang et al.,

1999b ). Thus the CaR is a candidate for mediating, at least

in part, the previously described direct actions of Cao21 on

chondrocytes and cartilage growth.

In fact, elevating Cao21 exerts several direct effects

on RCJ3.1C5.18 cells, namely, dose dependently reduc-

ing the levels of the mRNAs that encode a major pro-

teoglycan in cartilage, aggrecan, the α 1 chains of type II

and X collagens, and alkaline phosphatase ( Chang et al.,

1999a ). In addition, treatment of this cell line with a CaR

antisense oligonucleotide for 48 to 72 hours reduced the

level of CaR protein expression significantly, in associa-

tion with enhanced expression of aggrecan mRNA ( Chang

et al., 1999a ), suggesting a mediatory role of the CaR in

regulating this gene. This same group has recently shown

that chondrocytes isolated from CaR knockout mice retain

responsiveness to Cao21 . However, this residual activity

may result from the fact that these mice are able to splice

out exon 5, which was inactivated by insertion of the neo-

mycin gene, to yield an in-frame, modestly truncated recep-

tor that may have residual biological activity ( Rodriguez

et al., 2005 ). These results demonstrate, therefore, that (1) Cao

21 modulates the expression of several important genes

in chondrocytic cells, (2) chondrocytic cells express CaR

mRNA and protein, and (3) the CaR mediates at least some

of these actions of Cao21 in this cell type. Thus the CaR

could potentially not only modulate bone turnover and / or

the coupling of bone resorption to its later replacement by

osteoblasts through its actions on bone cells and/or their

precursors, but might also participate in the control of skel-

etal growth through its effects on chondrocytes.

THE CaR AND INTEGRATION OF CALCIUM

AND WATER METABOLISM

In addition to its roles in tissues involved directly in Cao21

homeostasis, increasing evidence indicates that the CaR

is located in other cells and tissues where it contributes

to integrating the functions of distinct homeostatic sys-

tems, as discussed in this section. An illustrative example

is the capacity of the CaR to integrate certain aspects of

mineral and water metabolism. Some hypercalcemic

patients exhibit reduced urinary concentrating capacity

and, occasionally, frank nephrogenic diabetes insipidus

( Gill and Bartter, 1961 ; Suki et al., 1969 ). In addition, sim-

ply the presence of hypercalciuria, without accompanying

hypercalcemia, can impair urinary concentrating ability, as

illustrated by the enuresis present in some hypercalciuric

children, which resolves with dietary calcium restriction

and resolution of the hypercalciuria (Valenti et al., 2002) .

The presence of the CaR in several segments of the neph-

ron that participate in regulating urinary concentration

( Riccardi et al., 1998 ; Sands et al., 1997 ) has suggested

a novel mechanism(s) underlying the long-recognized

but poorly understood inhibitory actions of high Cao21

on renal-concentrating capacity. Studies have shown that

high Cao21 , probably by activating CaRs residing on the

apical membrane of cells of the IMCD, reversibly inhib-

its vasopressin-elicited water flow by about 35% to 40%

in perfused rat IMCD tubules ( Sands et al., 1997 ). Indeed,

the CaR is present in the same apical endosomes that con-

tain the vasopressin-regulated water channel, aquaporin-2

( Sands et al., 1997 ). This observation indicates that the

receptor potentially reduces vasopressin-enhanced water

flow in the IMCD by either promoting the endocytosis or,

more likely, blocking the exocytosis of these endosomes

( Procino et al., 2004 ). Furthermore, induction of chronic

hypercalcemia in rats through treatment with vitamin D

reduces the level of expression of the aquaporin-2 pro-

tein but not its mRNA ( Sands et al., 1998 ), which would

further decrease vasopressin-activated water flow in the

terminal-collecting duct. In addition to the mechanisms

just described, high Cao21 -elicited, CaR-mediated reduc-

tion in the reabsorption of NaCl in the MTAL ( Wang et al.,

1996, 2001 ) , by decreasing the medullary countercurrent

gradient, would diminish even further the maximal urinary-

concentrating power of hypercalcemic patients ( Figure 3 ).

CaR-evoked generation of prostaglandins, especially PGE 2 ,

due to upregulation of Cox-2 in MTAL, may impair NaCl

reabsorption in this nephron segment and, in turn, genera-

tion of the hypertonic interstitium needed for normal uri-

nary concentrating ability ( Wang et al., 2001 ).

What is the evidence that these effects of high Cao21

on the renal concentrating mechanism are mediated by

the CaR? Of interest in this regard, persons with inacti-

vating mutations of the CaR are capable of concentrating

their urine normally despite their hypercalcemia ( Marx

et al., 1981 ), presumably because they are “ resistant ” to the

usual suppressive effects of high Cao21 on the urinary-

concentrating mechanism. Conversely, individuals with

activating CaR mutations can develop symptoms of dimin-

ished urinary-concentrating capacity (e.g., polyuria and

polydipsia), even at normal levels of Cao21 , when treated

with vitamin D and calcium supplementation, probably

because their renal CaRs are overly sensitive to Cao21

( Pearce et al., 1996 ). These experiments in nature, there-

fore, support the postulated, CaR-mediated link between

Cao21 and water homeostasis.

Is the defective renal handling of water in hypercal-

cemic patients of any physiological relevance? We have

suggested previously that it provides a mechanism that

integrates the renal handling of divalent cations, especially


Ca 2 1 , and water, thereby allowing appropriate “ trade-offs ”

in how the kidney coordinates calcium and water metabo-

lism under specific physiological conditions ( Hebert et

al., 1997 ; see Fig. 3 ). For example, in a situation where a

systemic load of Ca 2 1 , must be disposed of, the resultant

CaR-mediated inhibition of PTH secretion and direct inhi-

bition of tubular reabsorption of Ca 2 1 , promote calciuria.

The consequent elevation in luminal Cao21 in the IMCD,

particularly in a dehydrated individual, could predispose

to the formation of Ca 2 1 -containing renal stones, were not

for the concomitant, CaR-mediated inhibition of maximal

urinary concentration. Furthermore, there are abundant

CaRs in the subfornical organ (SFO; Rogers et al.,

1997 ) — an important hypothalamic thirst center ( Simpson

and Routenberg, 1975 ) — that may ensure a physiologically

appropriate increase in drinking. This increased intake of

free water could prevent dehydration that might otherwise be

a consequence of renal loss of free water due to the concom-

itant, CaR-induced inhibition of the urinary-concentrating

mechanism (see Fig. 3 ).

Finally, available data support the existence of a

“ calcium appetite ” in rats ( Tordoff, 1994 ) that may fur-

nish a mechanism for modulating the intake of calcium-

containing foods in a physiologically relevant manner

Increased Ca21

and Mg21

excretion in amore dilute urine

Decreased gutmotility, increasedH2O absorption

Thirst, H2Odrinking

Increased urinaryCa21 excretion

without riskof stones

Decreased urinaryconcentration

Decreased Ca21

reabsorption, increased

Ca21 excretion

Ca21 and Mg21

reabsorptionUrinary

concentratingability

Countercurrentmultiplication

H2O Reabsorption bycollecting duct

Ca21-receptoractivation

NaCI reabsorptionin TAL

Lumenpositive voltage

Hypercalcemiahypermagnesemia

FIGURE 3 Mechanisms that may interrelate systemic Cao

21 and water homeostasis (see text for further details). ( Top ) Mechanisms through which

the CaR reduces maximal urinary-concentrating ability. Reproduced with permission from Brown and Hebert (1997b) . ( Bottom ) Additional extrarenal

mechanisms integrating Cao21 and water homeostasis, such as

Cao

21 -evoked activation of the CaR in the SFO, which would enhance water intake and

mitigate loss of free water that would otherwise result from a diminished maximal urinary-concentrating capacity. Reproduced with permission from

Brown et al . (1996) .

Cao21


during hypo- and hypercalcemia. Some reduction in the

intake of Cao21 -containing foods might also occur as a

result of the activation of CaRs in the area postrema of the

brain — a “ nausea center ” ( Rogers et al., 1997 ) — due to

the resultant anorexia/nausea. Calcium appetite, in con-

trast, appears to be mediated by the subfornical organ

( McCaughey et al., 2003 ). The role of the CaR in either

of these actions, however, is currently conjectural. We have

hypothesized, therefore, that the CaR mediates multiple

layers of integration and coordination of the homeostatic

systems governing water and calcium metabolism. In doing

so, it may contribute to the ability of terrestrial organisms

to adjust to the only intermittent availability of environ-

mental calcium and water ( Hebert et al., 1997 ).

CaR-mediated modulation of vasopressin-induced

water flow in the IMCD represents an example of “ local ”

Cao21 homeostasis. That is, Cao

21 within a specific micro-

environment, which is outside of the blood and the various

compartments of the ECF in direct contact with the circula-

tion, is only allowed to rise to a certain level ( Brown et al.,

1999 ). Interestingly, changes in the level of Cao21 resulting

from the mechanism governing systemic Cao21 homeosta-

sis are traditionally thought to occur through fine adjust-

ments of the movements of Ca 2 1 into or out of the ECF

(i.e., by intestine, bone, or kidney; Brown, 1991 ). In con-

trast, CaR-mediated regulation of Cao21 in the IMCD pri-

marily results from alterations in the movement of water.

Perhaps even this formulation is oversimplified. On the one

hand, vasopressin is known to increase distal tubular reab-

sorption of calcium ( Hoenderop et al., 2000 ), which would

also reduce the level of Cao21 in the collecting duct. On

the other, the increased thirst in hypercalcemic patients, in

addition to providing more free water so as to mitigate any

associated rise in Cao21 in the IMCD, would also dilute

Cao21 in the ECF. Further studies will no doubt illuminate

additional subtleties related to how the body integrates

divalent mineral and water metabolism.

OTHER CaR AGONISTS AND

MODULATORS: THE CaR AS AN

INTEGRATOR OF PHYSIOLOGICAL

SIGNALS AND AS A “ NUTRIENT SENSOR ”

A variety of divalent cations ( Sro21 ), trivalent cations (e.g.,

Gdo31 ) , and even organic polycations (i.e., spermine; Quinn

et al., 1997 ) are effective CaR agonists. It is likely that they

all interact with one or more binding sites within the ECD

of the receptor ( Brown et al., 1999 ). Only a few of these

polycationic agonists, however, are thought to be present

within biological fluids at levels that would activate the

CaR ( Quinn et al., 1997 ). In addition to Cao21 , Mgo

21 and

spermine are two such putative, physiological CaR ago-

nists. It is probable that in specific microenvironments [e.g.,

within the gastrointestinal (GI) tract and central nervous

system] the concentrations of spermine are high enough to

activate the CaR even at levels of Cao21 that are insufficient

to do so by themselves ( Brown et al., 1999 ; Quinn et al.,

1997 ). In fact, all of the polycationic CaR agonists potenti-

ate one another’s stimulatory effects on the receptor. In other

words, only small increments in the level of a given agonist

(i.e., spermine) may be sufficient to activate the CaR when

a threshold level of another agonist is present in the local

microenvironment (e.g., Cao21 ; Brown et al., 1999 ).

Is the CaR also a Mgo21 -sensing receptor? Some evi-

dence supporting the role of the CaR in sensing and,

therefore, “ setting ” Mgo21 comes from the experiments in

nature that firmly established the role of the CaR as a cen-

tral element in Cao21 homeostasis. Namely, persons with

hypercalcemia due to heterozygous-inactivating mutations

of the CaR (e.g., FHH) exhibit serum Mg 2 1 levels that are

in the upper part of the normal range or mildly elevated.

Moreover, some patients with neonatal severe hyperpara-

thyroidism due to homozygous inactivating CaR muta-

tions can have more pronounced hypermagnesemia ( Aida

et al ., 1995a ). Conversely, persons harboring activating

mutations of the CaR can manifest mild reductions in

Mgo21 ( Brown, 1999 ). Mgo

21 is about twofold less potent

than Cao21 on a molar basis in activating the CaR ( Brown

et al., 1993 ; Butters et al., 1997 ). One might justifiably

ask, therefore, how Mgo21 could regulate its own homeo-

stasis by modulating PTH secretion — an important compo-

nent of CaR-mediated control of Cao21 — when circulating

levels of Mgo21 are, if anything, lower than those of Cao

21

( Bringhurst et al., 1998 )? It is possible that even small

changes in Mgo21 can modulate the activity of the CaR in

parathyroid cells because the receptor has been sensitized

by ambient levels of Cao21 that are close to its “ set point ”

(i.e., on the steepest portion of the curve relating PTH to

Cao21 ). A more likely scenario, however, is that acts on the

CaR in the CTAL to regulate its own level in the ECF, as

follows: The fraction of Mg 2 1 reabsorbed in the proximal

tubule is less than for other solutes (e.g., Ca 2 1 , Na 1 , Cl – ,

and water). As a result, there is a 1.6- to 1.8-fold rise in

the level of Mgo21 , in the tubular fluid of the CTAL ( De

Rouffignac and Quamme, 1994 ), which should, therefore,

reach a sufficiently high level to activate the CaR in this

nephron segment and, therefore, reduce the reabsorption of

Mgo21 . Recall that not only Cao

21 but also Mgo21 inhibits

the reabsorption of both divalent cations in perfused CTAL

( De Rouffignac and Quamme, 1994 ).

Another factor modulating the actions of Cao21 and

other polycations on the CaR is ionic strength per se

(e.g., alterations in the concentration of NaCl; Quinn

et al., 1998 ). Elevating ionic strength decreases and reduc-

ing ionic strength enhances the sensitivity of the CaR

to activation by Cao21 and Mgo

21 . The impact of chang-

ing ionic strength on the responsiveness of the CaR to

divalent cations may be especially relevant in particular

microenvironments, such as the GI or urinary tracts, where


ionic strength can vary greatly, easily encompassing the

range over which this parameter modulates the function of

the receptor ( Quinn et al., 1998 ).

In addition to the polycationic CaR agonists just noted,

novel “ calcimimetic, ” allosteric activators of the receptor

have been developed. These are small hydrophobic mol-

ecules, which are derivatives of phenylalkylamines and

activate the CaR by increasing its apparent affinity for

Cao21 . They do so by interacting with the transmembrane

domains of the receptor ( Nemeth et al., 1998b ), in con-

trast to Cao21 which binds to the ECD ( Hammerland et al.,

1999 ). Calcimimetics are called “ modulators ” rather than

“ agonists ” because they only activate the CaR in the pres-

ence of Cao21 . In contrast, the polycationic agonists of the

CaR (e.g., Gdo31 ) activate it even in the nominal absence

of Cao21 ( Nemeth et al., 1998b ). Calcimimetics have been

approved for use in patients with stage 5 chronic kidney

disease (i.e., on dialysis) and those with parathyroid cancer,

where they provide an effective medical means of lowering

PTH secretion through their direct action on the parathy-

roid CaR. A detailed discussion of the therapeutic utility of

these agents is beyond the scope of this review, but detailed

descriptions of their mechanism of action and clinical

use can be found in recent reviews ( Nemeth, 2004 ). CaR

antagonists, so-called “ calcilytics, ” are also entering clini-

cal trials. The principal therapeutic application envisioned

for these agents at the moment is in the treatment of osteo-

porosis ( Nemeth, 2004 ). Because intermittent exogenous

administration of PTH can produce sizable increases in

bone mineral density, once-daily administration of a short-

acting calcilytic would presumably accomplish the same

goal by producing a “ pulse ” of endogenous PTH secretion

( Gowen et al., 2000 ; Nemeth et al., 1998a ).

Amino acids represent another class of endogenous CaR

modulators ( Conigrave et al., 2000 ; Figure 4 ). Activation of

the CaR by specific amino acids, particularly the aromatic

amino acids, phenylalanine, tyrosine, histidine, and trypto-

phan, only occurs when Cao21 is 1 m M or higher. Although

individual amino acids are of relatively low potency (e.g.,

they act at concentrations of 0.1 to 1 m M or higher), a mix-

ture of amino acids with a composition similar to that pres-

ent in human serum under fasting conditions substantially

enhances the sensitivity of the CaR to Cao21 . That is, optimal

concentrations of the mixture reduce the EC 50 for Cao21 (the

level of Cao21 half-maximally activating the receptor) by

nearly 2 m M (e.g., by 40% to 50%) in HEK293 cells stably

transfected with the CaR ( Conigrave et al., 2000 ). Amino

acids also inhibit PTH secretion in vitro from human para-

thyroid cells, further supporting their biological relevance as

CaR activators ( Conigrave et al., 2004 ).

Although the implications of these direct actions of

amino acids on the CaR are not yet clear, they could poten-

tially explain several long-standing but poorly under-

stood observations appearing to link protein and Cao21

metabolism. Additionally, they suggest future lines of

research directed at elucidating the possible role of the

receptor in “ nutrient-sensing ” more generally, rather than

just as a sensor of divalent cations. For example, ingestion

of a high-protein diet nearly doubles the rate of urinary

calcium excretion relative to that observed on a low pro-

tein intake ( Insogna and Broadus, 1987 ). This effect of

dietary protein has traditionally been ascribed to the buff-

ering of acidic products of protein metabolism by bone as

well as a calciuric action of the acid load ( Lemann et al.,

1966 ). However, direct activation of CaRs in the CTAL as

a result of increases in serum levels of amino acids might

contribute as well. Moreover, reducing dietary protein

intake has been shown to have marked effects on circulat-

ing calciotropic hormones, nearly doubling serum PTH

and 1,25(OH) 2 D 3 levels in normal women ( Kerstetter

et al., 1997 ). Could these latter changes result from the

concomitant decrease in the circulating levels of amino

acids, despite little, if any, change in Cao21 ? Is it possible

0.30

0.25

0.2

0.15

0.1

0.05

0

0.25

0.2

0.15

0.1

0.05

0

0.03

0 1 2 3 4 5

0.1 1.0 10 100

D-Phe

L-Phe

Phenylalanine concentration (mM)

L-amino acid mixture (Fold concentration)

Ch

an

ge

in

flu

ore

sce

nce

ra

tio

(D3

40

/38

0)

Ch

an

ge

in

flu

ore

sce

nce

ra

tio

(D3

40

/38

0)

Ca21 1.0 mM

Ca21 1.5 mM

Ca21 2.5 mM

FIGURE 4 Amino acid sensing by the CaR. ( Top ) Activation of the

CaR by phenylalanine (l-phenylalanine . D-phenylalanine) at 2.5 m M

Cao21 in HEK293 cells transfected stably with the CaR as reflected by

amino acid-induced increases in the cytosolic Cao21 concentration in

cells loaded with the Cao21 -sensitive intracellular dye fura-2. Increases

in the fluorescence ratio (340/380 nm) indicate CaR-mediated increases in

Ca i

21 . ( Bottom ) Marked impact on the level of Cao

21 on the capacity of

the CaR to sense the mixture of l-amino acids that emulates that present

in the blood under fasting conditions (see text for details). Reproduced

with permission from Conigrave et al . (2000) .


that the reduced intake of dietary protein commonly rec-

ommended for patients with chronic renal insufficiency

( Bringhurst et al., 1998 ) actually exacerbates their second-

ary hyperparathyroidism?

These observations suggest that the CaR is not solely

a Cao21 (and probably a Mgo

21 ) receptor but may also

serve as a more general “ nutrient ” and environmental sen-

sor, which detects changes in Cao21 and Mgo

21 , not in

isolation, but in the context of the ambient levels of cer-

tain amino acids. Further testing of this hypothesis may

enhance our understanding of the mechanisms by which

complex organisms coordinate homeostatic systems that

have traditionally been thought of as functioning largely

independently, such as those controlling protein and min-

eral metabolism. This homeostatic integration may be par-

ticularly important within specific parts of the life cycle,

such as during somatic growth. Skeletal growth in child-

hood requires the precisely coordinated deposition of both

bone matrix and mineral. Moreover, mineral ions and

amino acids must also be assimilated during the growth of

soft tissues — all of which contain varying mixtures of min-

eral ions and protein. For instance, smooth muscle cells

contain half as much calcium as bone when expressed on

the basis of wet weight ( Brown and MacLeod, 2001 ).

Coordinating mineral ion and protein metabolism might

be particularly relevant in the GI tract. Indeed, the pres-

ence of an “ amino acid receptor ” regulating the secretion

of gastrin, gastric acid, and cholecystokinin has been pos-

tulated ( Conigrave et al., 2000 ). Furthermore, the pharma-

cological profile of the actions of different amino acids on

these parameters is strikingly similar to that for the effects

of the same amino acids on the CaR ( Mangel et al., 1995 ;

McArthur et al., 1983 ; Taylor et al., 1982; Conigrave et al.,

2000 ). CaRs in the GI tract system could serve as a particu-

larly suitable target for sensing the availability of dietary

protein and mineral ions, which are generally ingested

together (i.e., in milk). Further studies are needed, there-

fore, to investigate whether the CaR represents, in fact,

this putative amino acid receptor. Such investigations may

reveal whether the sensing of amino acids by the CaR,

taken in the context of ambient levels of Cao21 and Mgo

21

within the GI tract and elsewhere, provides the molecular

basis for a physiologically important link between the sys-

tems governing protein and mineral metabolism.

ARE THERE ADDITIONAL Cao21 SENSORS?

As noted earlier and described in detail in other reviews

( Quarles, 1997 ; Zaidi et al., 1999 ; Pi et al., 2005 ), Cao21

sensors in addition to the CaR may exist on osteoblasts and

osteoclasts. Moreover, studies have revealed that some of

the mGluRs can sense Cao21 in addition to recognizing

glutamate as their principal physiological agonist, although

the physiological importance of this Cao21 sensing is not

clear at present. Kubo et al. (1998) showed that mGluRs

1, 3, and 5 sense levels of Cao21 between 0.1 and 10 m M,

whereas mGluR2 is substantially less responsive to Cao21 .

All three of the mGluRs that are capable of sensing Cao21

have identical serines and threonines, respectively, at amino

acid positions that are equivalent to amino acid residues 165

and 188 in mGluR1a ( Brauner-Osborne et al., 1999 ). These

two residues have been shown to play key roles in the bind-

ing of glutamate to the respective ECDs of the MGluRs

( O’Hara et al., 1993 ). In contrast, whereas mGluRs 1a, 3,

and 5 have serines at positions equivalent to amino acid

residue 166 in mGluR1a, mGluR2 has an aspartate rather

than a serine at this position ( Kubo et al., 1998 ). Changing

this serine to an aspartate in mGluRs 1a, 3, and 5 substan-

tially reduces their capacities to sense Cao21 , whereas sub-

stituting the aspartate in mGluR2 with a serine enhances its

apparent affinity for Cao21 to a level comparable to those

of mGluRs 1, 3, and 5 ( Kubo et al., 1998 ). Thus the serines

in mGluRs 3 and 5 at amino acid positions homologous to

residue 166 in mGluR1a appear to play important roles in

the capacities of these three receptors to sense Cao21 .

Interestingly, another study has shown that changes in

Cao21 also modulate the function of the activated GABA B

receptors, whereas Cao21 has no effect on these receptors

in the absence of added GABA ( Wise et al., 1999 ). Cao21

potentiates the stimulatory effect of GABA on the binding

of GTP to the receptor and increases the coupling of the

GABA B receptor to stimulation of a K 1 channel and inhibi-

tion of forskolin-stimulated adenylate cyclase activity. The

effects of Cao21 on the GABA B receptor, unlike those on

the CaR, were not reproduced by other polyvalent cations.

Thus, similar to the CaR, which senses Cao21 but is modu-

lated by various amino acids (although not by glutamate;

Conigrave et al., 2000 ), mGluRs and GABA B receptors

sense their primary physiological ligands, glutamate and

GABA, respectively, as well as Cao21 . These observations

further emphasize the structural, functional, and evolution-

ary relationships among the three types of receptors.

Finally, Cao21 could also, of course, modulate the

functions of proteins other than GPCRs. For instance,

the recently cloned Cao21 channels TRPV5 and 6 can be

viewed as operating on a macroscopic level as facilitated

transporters. That is, they exhibit Michaelis–Menten-like

kinetics, their activities (measured as 45 Ca 2 1 uptake in X.

laevis oocytes) increase with the level of Cao21 until Ca 2 1

uptake saturates above about 1 m M Cao21 . They could

potentially function, therefore, as Cao21 sensors. That is,

they would tend to “ set ” the level of Cao21 within the local

ECF by increasing Ca 2 1 uptake when Cao21 is high and

reducing it when it is low.

SUMMARY

The discovery of the CaR has provided a molecular mecha-

nism that mediates many of the known actions of Cao21 on

the cells and tissues that participate directly in systemic

Cao21 homeostasis, such as parathyroid and certain renal


cells. There is still much to be learned, however, about the

various functions of the receptor in these tissues, particu-

larly in intestinal and bone cells, as well as in the numerous

other CaR-expressing cells that are not directly involved in

systemic Cao21 homeostasis. In these latter, “ nonhomeo-

static ” cells, the CaR probably serves a variety of roles that

enable it to serve as a versatile first messenger, capable of

regulating numerous cellular functions. Moreover, the abil-

ity of the CaR to integrate and coordinate several different

ionic and nutritional signals may permit it to act as a cen-

tral homeostatic element not only for mineral ion homeo-

stasis, but also for processes relevant to Mgo21 , water, and

protein metabolism.

ACKNOWLEDGMENTS

The author gratefully acknowledges generous grant support

from the NIH (DK52005, DK67111, and DK67155).

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