HORMONAL REGULATION OF CALCIUM HOMEOSTASIS

Overview of integrated regulation of calcium homeostasis

Tight regulation of the ECF calcium concentration is maintained through the action of Ca‐sensitive cells which modulate the production of hormones [2–5]. These hor­

mones act on specific cells in bone, gut, and kidney which can respond by altering fluxes of Ca to maintain ECF Ca.

Thus a reduction in ECF Ca stimulates release of PTH from the parathyroid glands in the neck (Fig. 22.2). This hormone can then act to enhance bone resorption and lib­

erate both Ca and phosphate from the skeleton. PTH has also been reported to increase release of the phosphaturic hormone, FGF23, from mature osteoblasts and osteocytes [6]. PTH can augment Ca reabsorption in the kidney and at the same time reduce phosphate reabsorption produc­

ing phosphaturia. Hypocalcemia and  PTH itself can both stimulate the conversion of the inert metabolite of vitamin D, 25‐hydroxyvitamin D (25OHD), to the active moiety 1,25‐dihydroxyvitamin D [1,25(OH)₂D] [7] which in turn will augment intestinal Ca absorption, and to a lesser extent renal phosphate reabsorption. The net effect

Intestine Bone

Ca 1.0 g/day

Ca 0.2 g/day

Ca 0.5 g/day

Ca 1.0 kg

Ca 10.0 g/day

Urine Ca 0.2 g/day Ca 9.8 g/day

Ca 0.8 g/day

Kidney

ECF Ca

Fig. 22.1. Calcium balance. On average, in a typical adult, approximately 1 g of elemental calcium (Ca²⁺) is ingested per day. Of this, about 200 mg/day will be absorbed and 800 mg/day excreted. Approximately 1 kg of Ca is stored in bone and about 500 mg/day is released by resorption and deposited during bone formation when bone turnover is in balance. Of the 10 g of Ca filtered through the kidney per day only about 200 mg or less appears in the urine, the remainder being reabsorbed.

of the mobilization of Ca from bone, the increased absorp­

tion of Ca from the gut, and the increased reabsorption of filtered Ca along the nephron is to restore the ECF Ca to  normal and to inhibit further production of PTH and  1,25(OH)₂D. Furthermore, released FGF23 can also reduce 1,25(OH)₂D [8] and has been reported to decrease PTH production therefore further ensuring that Ca homeostasis is restored.

When the ECF Ca is raised above the normal range, the opposite sequence of events occurs, ie, diminished PTH secretion caused by stimulation of the parathyroid Ca‐

sensing receptor (CaSR) and diminished renal 1,25(OH)₂D production. In addition, a direct calciuric effect of hyper­

calcemia on the kidney can occur mediated by the renal CaSR in the cortical thick ascending limb (CTAL) of Henle’s loop. Therefore, the effect of suppressing the release of PTH and production of 1,25(OH)₂D and of stim­

ulating renal CaSR results in diminished skeletal Ca release, decreased intestinal Ca absorption, and reduced renal tubular Ca reabsorption respectively, and restores the elevated ECF Ca to normal.

Regulation of hormone production

PTH production

A major regulator of parathyroid gland secretion of PTH is ECF Ca. The parathyroid glands detect ECF Ca via a CaSR, which is a Gq protein‐coupled receptor [9] Thus a decrease in ECF Ca is sensed by the CaSR in parathyroid

chief cells resulting in an acute increase in PTH secre­

tion. The relationship between ECF Ca and PTH secre­

tion is governed by a steep inverse sigmoidal curve which is characterized by a maximal secretory rate at low ECF Ca, a midpoint or "set point" which is the level of ECF Ca which half‐maximally suppresses PTH, and a minimal secretory rate at high ECF Ca [10, 11]. Sustained hypocal­

cemia can eventually lead to parathyroid cell prolifera­

tion [12] and an increased total secretory capacity of the parathyroid gland. ECF Ca, acting via the CaSR, therefore functions as a hormone in modulating PTH release and parathyroid cell function. 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) reduces PTH synthesis and parathyroid cell proliferation [13]. Molecular events in PTH secretion and CaSR function are found in Chapters 26 and 28.

Vitamin D production and metabolism

Vitamin D produced in the skin or ingested in the diet is 25‐hydroxylated in liver and the 25OHD metabolite thus  formed is then converted in the kidney and, to a lesser extent, in other tissues [14] to the active form, 1,25(OH)₂D, by a mitochondrial enzyme, 25OHD‐1α hydroxylase [CYP27B1 or 1α(OH)ase] The renal pro­

duction of 1,25(OH)₂D is stimulated by hypocalcemia, hypophosphatemia, and elevated PTH levels. The renal 1α(OH)ase is potently inhibited by 1,25(OH)₂D as part of a negative feedback loop. Furthermore, 1,25(OH)₂D can also stimulate a renal 24‐hydroxylase enzyme [CYP24A1 or 24(OH)ase], converting 25OHD into 24,25(OH)₂D, and 3

9

2 1 7

5 10

5 8 6

4

↑Ca⁺⁺

Ca⁺⁺

CaSR

CaSR

11

1,25(OH)₂D 25(OH)D

FGF23 PTH

Fig. 22.2. Hormonal regulation of ECF Ca homeostasis. Decreased (↓) ECF Ca results in increased PTH release from the parathyroid glands via the CaSR (1). The increased PTH can enhance Ca reabsorption from the kidney (2), and with decreased ECF Ca, CaSR is not activated to cause calciuria. PTH can also increase conversion of 25OHD to 1,25(OH)₂D in the kidney (3). The 1,25(OH)₂D produced can increase intestinal absorption of Ca (4), and PTH and 1,25(OH)₂D can resorb bone and increase Ca release from bone (5). The net result is normalization (→) of ECF Ca and inhibition (┤) of further PTH release (7). 1,25(OH)₂D can also stimulate FGF23 release from bone (8) which in turn can inhibit further renal 1,25(OH)₂D production from 25OHD (9). PTH may also stimulate FGF23 (10) which may then limit further PTH release (11).

168 Regulation of Calcium Homeostasis

thereby reducing the substrate of the renal 1α(OH)ase, and converting 1,25(OH)₂D into 1,24,25(OH)₃D, which is then metabolized to calcitroic acid (1‐hydroxy‐23‐

carboxy‐vitamine D3) or 23,25OHD‐26,23‐lactone, two inactive forms of vitamin D. FGF23 is a potent inhibitor of the renal 1α(OH)ase and can also stimulate the renal 24(OH)ase thereby participating in the reduction of circu­

lating 1,25(OH)₂D concentrations. In this way, FGF23 acts as a counter regulatory hormone for 1,25(OH)₂D effects on mineral homeostasis. The molecular details of  the vitamin D metabolic pathway are described in Chapter 29.

FGF23 production

FGF23 may be stimulated by both local and systemic fac­

tors. In humans, high dietary phosphorus increases and low dietary phosphorus decreases serum FGF23 [15–18];

but the changes are modest, and there is a lag period between phosphate loading and elevations of FGF23 [17].

Serum Ca‐mediated increases in serum FGF23 were shown, in mice, to require a threshold level of serum phosphorus and, likewise, phosphate‐elicited increases in FGF23 were markedly blunted if serum Ca was below a threshold level [19]. PTH can increase FGF23 but ambi­

ent concentrations of 1,25(OH)2D appear to supersede the effects of PTH on regulating this hormone [20]. Thus 1,25(OH)₂D appears to be the most important physiologi­

cal stimulus for FGF23 production and FGF23 and 1,25(OH)₂D participate in a bone–kidney endocrine loop, in which 1,25(OH)₂D stimulates FGF23 production and FGF23 suppresses 1,25(OH)₂D levels.

PTH, 1,25(OH)

₂

D

_,

calcium, and FGF23 actions in target tissues to regulate calcium homeostasis

Intestinal Ca transport

Net intestinal Ca absorption can be determined by the external balance technique in which a diet of known composition with a known amount of Ca is ingested, and urine Ca excretion and fecal Ca loss are measured.

Negative balance occurs when net absorption declines to about 200 mg Ca per day (5.0 mmol). The portion of die­

tary Ca absorbed varies with age and amount of Ca ingested, and may range from 20 to 60%. Rates of net Ca absorption are high in growing children, during growth spurts in adolescence, and during pregnancy and lacta­

tion. The efficiency of Ca absorption increases during prolonged dietary Ca restriction to absorb the greatest portion of that ingested. Net absorption declines with age in men and women, and so increased Ca intake is required to compensate for the lower absorption rate. Fecal Ca losses vary between 100 and 200 mg per day (2.5 to 5.0 mmol). Fecal Ca is composed of unabsorbed dietary Ca and Ca contained in intestinal, pancreatic, and biliary secretions. Secreted Ca is not regulated by hormones or serum Ca.

About 90% of absorbed Ca occurs in the large surface area of the duodenum and jejunum. Increased Ca require­

ments stimulate expression of the epithelial Ca active transport system in duodenum, ileum, and colon sufficient to increase fractional Ca absorption from 20% to 45% in older men and women and to 55% to 70% in children and young adults. 1,25(OH)₂D₃ increases the efficiency of the gut to absorb dietary Ca. Intestinal epithelial Ca transport includes both an energy‐dependent, cell‐mediated satura­

ble active process that is largely regulated by 1,25(OH)₂D, and a passive, diffusional paracellular path of absorption that is driven by transepithelial electrochemical gradients.

Active Ca absorption accounts for absorption of 10% to 15% of a dietary load [21]. Active transcellular intestinal absorption involves three sequential cellular steps: first, a rate‐limiting step involving transfer of luminal Ca into the intestinal cell, via the epithelial apical Ca channel of the transient receptor potential vanilloid (TRPV) family, TRPV6; second, transport of Ca across the cell via a chan­

nel‐associated protein, calbindin‐D9K; and finally extru­

sion of Ca across the basolateral membrane into ECF by an energy‐requiring process via the basolateral Ca ATPase system, PMCA1b [21, 22]. Reductions in dietary Ca intake can increase PTH secretion and 1,25(OH)₂D production.

Increased 1,25(OH)₂D can then increase expression of these proteins resulting in enhanced fractional Ca absorption and compensation for the dietary reduction [23] (see Chapter  29). This cell‐mediated pathway involving the TRPV6 Ca channel is saturable with a K_t (1/2 maximal transport) of 1.0 mM.

By regulating claudin 2 and claudin 12, which form paracellular calcium channels, 1,25(OH)₂D may also reroute Ca through paracellular epithelial cell junctions.

However, passive diffusion increases linearly with luminal Ca concentration and during high dietary Ca intake, 1,25(OH)₂D is suppressed and passive paracellular transport accounts for almost all absorption. Causes of increased and decreased intestinal Ca absorption are listed in Table 22.1.

Renal Ca handling

The kidney plays a central role in ensuring Ca balance, and PTH has a major role in fine‐tuning this renal function [24–26] along with ECF Ca per se. Multiple influences on Ca handling are listed in Table  22.2 (Chapters 26,28, and 29 contain descriptions of the molecular actions of PTH on the kidney). PTH has little  effect on modulating Ca fluxes in the proximal tubule where 65% of the filtered Ca is reabsorbed, coupled to the bulk transport of solutes such as sodium and water [25]. In this nephron region, PTH can stimu­

late the 1α(OH)ase, leading to increased synthesis of 1,25(OH)₂D [27]. A reduction in ECF Ca can itself stimu­

late 1,25(OH)₂D production but whether this occurs via the CaSR is presently unknown. Finally PTH can also inhibit Na and HC0_3‐ reabsorption in the proximal tubule by inhibiting the apical type 3 Na⁺/H⁺ exchanger [28], and the basolateral Na⁺/K⁺‐ATPase [29], and can

inhibit apical Na+/PO₄‐ cotransport by inhibiting the type II Na + ‐dependent phosphate cotransporters NaPi‐

IIa and NaPi‐IIc.

About 20% of filtered Ca is reabsorbed in the cortical thick ascending limb of the loop of Henle (CTAL) and 15%

is reabsorbed in the distal convoluted tubule (DCT). At both sites PTH binds to the PTH receptor (PTHR) [30, 31], and enhances Ca reabsorption. In the CTAL, at least, this appears to occur by increasing the activity of the Na/K/2Cl cotransporter that drives NaCl reabsorption and stimu­

lates paracellular Ca and Mg reabsorption [32].

The CaSR is also resident in the CTAL [33], where increased ECF Ca activates phospholipase A2, thereby reducing the activity of the Na/K/2Cl cotransporter and of an apical K channel, and diminishing paracellular Ca reabsorption. Consequently, a raised ECF Ca antagonizes the effect of PTH in this nephron segment and ECF Ca can in fact participate in this way in the regulation of its own homeostasis. Inhibition of NaCl reabsorption and loss of NaCl in the urine may contribute to the volume depletion observed in severe hypercalcemia. ECF Ca may therefore act in a manner analogous to “loop” diuretics

such as furosemide. 1,25(OH)₂D₃ has a direct effect on  renal Ca handling through stimulation of CaSR. It remains controversial whether 1,25(OH)₂D₃ plays a direct role in enhancing tubular Ca reabsorption in humans although it appears to do so in mouse models.

In the DCT, PTH can also influence [21] luminal Ca trans­

fer into the renal tubule cell via TRPV5, translocation of Ca across the cell from apical to basolateral surface involving proteins such as calbindin‐D28K, and finally active extru­

sion of Ca from the cell into the blood via a Na+/Ca exchanger, designated NCX1. PTH markedly stimulates Ca reabsorption in the DCT primarily by augmenting NCX1 activity via a cyclic AMP‐mediated mechanism.

Bone remodeling and mineralization, and Ca homeostasis

In bone, the PTHR is localized on cells of the osteoblast phenotype which are of mesenchymal origin [34] but not on osteoclasts which are of hematogenous origin. A major physiological role of PTH appears to be to maintain nor­

mal Ca homeostasis by enhancing release of the cytokine, receptor activator of NFkB ligand (RANKL) [35]. RANKL binds to its receptor, RANK, on osteoclast precursors and osteoclasts, enhancing the formation of mature osteo­

clasts from precursors and increasing the resorptive activ­

ity of existing osteoclasts especially in cortical bone. PTH may also reduce the osteoblastic protein, osteoprotegerin, Table 22.1. Conditions that increase or decrease intestinal

Ca absorption.

Increased Ca Absorption Decreased Ca Absorption Increased renal 1,25(OH)₂D

production Decreased renal

1,25(OH)₂D production

Growth Vitamin D deficiency

Pregnancy(may also include increased placental production)

Chronic renal insufficiency Hypoparathyroidism Aging

25‐hydroxylase deficiency(CYP2R1 mutation)

Vitamin D‐dependent rickets

type I (hereditary pseudo‐

vitamin D deficient rickets)

(CYP27B1 mutation) Resistance to 1,25(OH)₂D Vitamin D‐dependent

rickets

type II (hereditary vitamin D resistant rickets)(VDR mutation) Normal 1,25(OH)₂D

production

Glucocorticoid excess Hyperthyroidism Lactation

Primary hyperparathyroidism Idiopathic hypercalciuria Phosphate‐wasting disorders,

such as those caused by NPT2a or NPT2c mutations

Decreased renal 1,25(OH)₂D metabolism

Idiopathic infantile

hypercalcemia (CYP24A1 mutations)

Increased extra‐renal 1,25(OH)₂D production Sarcoid and other

granulomatous diseases B‐cell lymphoma

Table 22.2. Hormones and conditions that regulate urine Ca excretion via increasing or decreasing glomerular filtration and/or tubular reabsorption.

Decreased Ca Excretion Increased Ca Excretion Decreased glomerular

filtration Increased glomerular

filtration

Hypocalcemia Hypercalcemia

Hypomagnesemia Renal insufficiency Increased tubular

reabsorption Decreased tubular

reabsorption

Hypocalcemia Hypercalcemia

ECF volume contraction ECF volume expansion Thiazide diuretics Loop diuretics

Phosphate administration Phosphate deprivation Metabolic alkalosis Metabolic acidosis PTH — as in

hyperparathyroidism and pseudohypoparathyroidism

Cyclosporin A Dent disease Bartter syndrome Autosomal dominant

hypocalcemia (activating CaSR

mutation) Parathyroid hormone‐related

peptide (PTHrP) Gitelman syndrome Familial hypocalciuric

hypercalcemia

(inactivating CaSR mutation)

170 Regulation of Calcium Homeostasis

which binds to RANKL, forming an inactive complex, and preventing it from binding to RANK, thus reducing osteo­

clastic activity [36, 37]. It has also been suggested that PTH can acutely release mineral at the bone surface in an osteoclast‐independent manner by modifying its solubility [38]. PTH may also have anabolic effects via its action on osteoblastic cells, mainly on trabecular bone.

Vitamin D is essential for normal mineralization of bone that may be caused by an indirect effect of enhanc­

ing intestinal calcium and phosphate absorption and maintaining these ions within a range that facilitates hydroxyapatite deposition in bone matrix. A major direct function of 1,25(OH)₂D on bone appears to be to enhance mobilization of Ca stores when dietary Ca is insufficient to maintain a normal ECF Ca [39]. As with PTH [40], 1,25(OH)₂D enhances osteoclastic bone resorption by binding to receptors on cells of the osteoblastic lineage and increasing the RANKL/OPG ratio to enhance the proliferation, differentiation, and activation of the osteo­

clastic system from its monocytic precursors [41]. High levels of 1,25(OH)₂D may also inhibit mineralization.

Endogenous and exogenous 1,25(OH)₂D₃ have also been reported to have an anabolic role in vivo [42, 43].

Bone formation and resorption are discussed in detail in Section I, and Chapters 25 to 29. The molecular basis for physiological and pathological states of bone turnover is detailed in Chapters 67, 70, 73–76 and 79 and in Section VI.

Temporal sequence of regulation of calcium homeostasis

The elevation in circulating PTH level in response to hypocalcemia enhances distal renal tubular Ca reabsorp­

tion within minutes. There may also be PTH‐independ­

ent “buffering” of ECF Ca by bone through incompletely understood mechanisms, perhaps involving the CaSR, that rapidly returns ECF Ca to its baseline value after induction of hypocalcemia [44]. Therefore, a short period of hypocalcemia may be corrected exclusively through increased renal conservation of Ca and mobilization of Ca from bone. PTH‐induced osteoclastic bone resorption likely occurs after several hours to days. PTH‐induced stimulation of renal synthesis of 1,25(OH)₂D from 25OHD requires several hours [45] and more prolonged hypocalcemia with more prolonged exposure to elevated PTH, may involve a 1,25(OH)₂D‐mediated augmentation of intestinal Ca absorption, as well as 1,25(OH)₂D‐medi­

ated release of Ca from bone.

Generally, an elevation in circulating PTH in response to hypocalcemia is adequate to restore normocalcemia within minutes to a few hours. There are various clinical conditions, however (eg, markedly low Ca intake or vita­

min D deficiency), in which greater and more prolonged increases in PTH levels are required to restore and maintain normocalcemia. This can be achieved through a temporal graded series of responses of the parathyroid glands to low ECF Ca and/or associated deficiency of 1,25(OH)₂D [46]. Thus, after the initial release of stored

PTH in response to hypocalcemia, which occurs within seconds and lasts for 60 to 90 min, there is decreased intracellular degradation of PTH within 20 to 30 min [47], enhanced PTH gene expression over hours and, eventu­

ally, increased parathyroid cell proliferation over weeks to months or more [48]. In this situation, large increases in circulating PTH and in parathyroid cellular mass can occur, e.g. in patients with chronic kidney disease and severe hyperparathyroidism.

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