Calcium plays a vital role in several biological processes including normal neuromuscular transmission, muscular contractility, cellular signaling, enzyme function, and blood coagulation. Hence, the appropriate control of calcium homeostasis is vital to the well-being of the organism. Severe calcium deficiency results in tetany and, in extreme cases, grand mal seizures. The deposition of excess calcium at ectopic sites occurs in several diseases such as nephrolithiasis, arteriosclerosis, valvular calcification, and calciphylaxis. Therefore, an appreciation of how calcium is normally absorbed, excreted, and regulated is important in understanding the pathophysiology of disease.
Calcium homeostasis is a tightly controlled process involving several tissues, hormones, and proteins. The hormones 1α,25-dihydroxyvitamin D (1α,25(OH) 2 D), parathyroid hormone (PTH), and calcitonin contribute to the regulation of calcium metabolism. These three hormones have specific roles in several tissues including the intestinal mucosa, bone, and renal tubular cells. They work in concert with each other through complex interactions to maintain extracellular fluid calcium concentrations in the normal range.
Keywords
calcium, 25 hydroxyvitamin D 3 , 1,25 dihydroxyvitamin D 3 , parathyroid hormone, calcitonin
Introduction
Calcium plays a vital role in several biological processes. It is an important constituent bone mineral, and is necessary for normal neuromuscular transmission, muscular contractility, cellular signaling, enzyme function, and blood coagulation. Hence, the appropriate control of calcium homeostasis is vital to the well-being of the organism. A persistently negative calcium balance results in hyperparathyroidism and bone demineralization and contributes to the pathogenesis of osteomalacia and the development of osteoporosis with attendant vertebral and appendicular fractures. Severe calcium deficiency results in tetany and, in extreme cases, grand mal seizures. The deposition of excess calcium at ectopic sites occurs in several diseases such as nephrolithiasis, arteriosclerosis, valvular calcification, and calciphylaxis. Therefore, an appreciation of how calcium is normally absorbed, excreted and regulated is important in understanding the pathophysiology of disease.
Calcium homeostasis is a tightly controlled process involving several tissues, hormones, and proteins. The hormones 1α,25-dihydroxyvitamin D (1α,25(OH) 2 D), parathyroid hormone (PTH), and calcitonin contribute to the regulation of calcium metabolism. These three hormones have specific roles in several tissues including the intestinal mucosa, bone, and renal tubular cells. They work in concert with each other through complex interactions to maintain extracellular fluid calcium concentrations in the normal range. In this chapter, we will discuss the physiological role of each of these hormones in calcium homeostasis.
Calcium Balance
Many tissues are dependent on the maintenance of extracellular calcium concentrations within the physiological range for proper function. If extracellular calcium concentrations are significantly altered or disrupted, tissue and organ dysfunction may result (see above). Bone is the major reservoir of calcium, containing about 99% of total body calcium stores, and provides a significant buffer to maintain extracellular calcium concentrations within the normal range if calcium intake declines or calcium losses occur. The maintenance of serum calcium concentrations in the face of significant overall deficits in calcium balance occurs at the expense of bone integrity. Serum calcium concentrations (range 8.9–10.1 mg/dL in adults) are age dependent, and small differences in circulating concentrations occur as a result of gender. Table 65.1 shows normal serum calcium concentrations in males and females measured at the Mayo Clinic. Serum calcium is comprised of protein-bound calcium (40%); complexed calcium, i.e., calcium complexed to ions such as citrate, sulfate, and phosphate (13%); and ionized calcium (47%). Total serum calcium concentrations are dependent upon circulating concentrations of albumin and to a smaller extent upon circulating concentration of globulins. The biologically active fraction of serum calcium is ionized (normal range 4.8–5.7 mg/dL in adults) and, at a normal serum pH of 7.4, is approximately 47% of total serum calcium. The percentage of ionized calcium changes with pH—an alkaline pH causing a reduction in free ionized calcium concentrations and an acid pH causing an increase in ionized calcium concentrations. The relationships between ionized calcium, total calcium, albumin and pH are defined by the following equation:
[ CaProt ] = { 0 . 2111 [ Alb ] } − { ( 0 . 42 ) [ A l b ] 47.3 ( 7 . 42 − pH ) } CaProt = protein − bound calcium , mmoles / L .
| Serum Calcium Concentrations | |||
|---|---|---|---|
| Males | Females | ||
| Age (years) | Concentration (mg/dL) | Age (years) | Concentration (mg/dL) |
| 1–14 | 9.6–10.6 | 1–11 | 9.6–10.6 |
| 15–16 | 9.5–10.5 | 12–14 | 9.5–10.4 |
| 17–18 | 9.5–10.4 | 15–18 | 9.1–10.3 |
| 19–21 | 9.3–10.3 | ≥19 | 8.9–10.1 |
| ≥22 | 8.9–10.1 | ||
| Age- and Sex-Specific Serum Ionized Calcium Concentrations | |||
| 1–19 | 5.1–5.9 | 1–17 | 5.1–5.9 |
| ≥20 | 4.8–5.7 | ≥18 | 4.8–5.7 |
A 1-gram/deciliter change in serum albumin concentration is associated with a 0.8-milligram/deciliter change in total serum calcium. Since globulins bind calcium less avidly than does albumin, a 1-gram/deciliter change in globulins results in a 0.16-milligram/deciliter change in total serum calcium. It should be noted that changes in the concentrations of serum proteins are generally not associated with changes in the percentage of ionized calcium present in the circulation. Also, it is worth remembering that the amount of calcium filtered at the glomerulus of the kidney is a sum of the ionized calcium concentration and the complex calcium concentration (approximately 60% of total serum calcium concentration).
Under normal circumstances of neutral calcium balance in the adult human, net intestinal absorption of calcium is equal to urinary calcium ( Figure 65.1 ). Calcium flux into and out of bone is well balanced with equal amounts of calcium being deposited and resorbed. Over a 24-hour period, a typical human adult ingests about 1000 mg of elemental calcium. Approximately 40% of ingested calcium is absorbed in the intestine and enters the bloodstream. Both active and passive processes are involved in the absorption of calcium from the intestine. When the intestinal lumen calcium concentrations are <10 millimoles/liter, active processes play a major role in calcium absorption. However, when calcium concentrations in the intestinal lumen exceed 10 millimoles/liter, passive processes become operative in the absorption of calcium. About 150 mg of calcium are excreted into the gastrointestinal tract each day, predominantly in pancreatic and intestinal secretions (“endogenous fecal calcium”), for a net calcium absorption of about 250 mg per day. The kidney filters about 9000 mg of calcium each day in the glomeruli and reabsorbs the majority of filtered calcium in the proximal and distal nephron, resulting in a net loss of about 250 mg from the kidney in the urine. A majority of calcium is reabsorbed in the proximal tubule along with sodium (approximately 70 to 85%). The remainder of filtered calcium is reabsorbed in the thick ascending limb of the loop of Henle and in the distal convoluted tubule, largely as a result of the activity of the sodium-calcium exchanger and plasma membrane calcium pump (PMCA). In states of calcium balance, urinary calcium approximates the amount of calcium absorbed in the intestine.
The following sections will describe how the vitamin D endocrine system, PTH, and calcitonin alter calcium homeostasis in various tissues including the intestine, kidney, and bone.
Vitamin D Endocrine System
Nomenclature
The synthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D, requires sequential metabolic processing of precursor sterols in several tissues such as the skin, liver and kidney. The term vitamin D refers to both vitamin D 2 and vitamin D 3 ( Figure 65.2 ). The metabolic processing of both these forms of vitamin D is similar for practical purposes in mammals, although vitamin D 2 is considerably less active in birds than is vitamin D 3 . For the purposes of this chapter, it is appropriate to assume that the metabolic transformations that occur with vitamin D 3 also occur with vitamin D 2 . Vitamin D 2 is derived from the plant sterol, ergosterol, whereas, vitamin D 3 is derived from 7-dehydrocholesterol, a byproduct of steroid metabolism. In non-vitamin D supplemented individuals, a majority of circulating vitamin D is in the form of vitamin D 3 or cholecalciferol. Medicinal vitamin D preparations available in the United States today may contain either vitamin D 2 or vitamin D 3 . Individuals taking large amounts of vitamin D 2 supplements have elevated concentrations of vitamin D 2 and its 25-hydroxylated metabolite, 25-hydroxyvitamin D 2 .
Formation of Vitamin D
In the early 1900s, Huldshinsky and Chick in Vienna showed that exposure of rachitic children to ultraviolet light cured their bone disease. Steenbock and Hart showed that ultraviolet irradiation of animals would put them into positive calcium balance. Later, Steenbock and Black clearly demonstrated that ultraviolet light-induced antirachitic activity in the fat-soluble portion of foods and skin. Hess and Weinstock made similar observations. Building on these observations, Askew and his coworkers isolated vitamin D 2 . Windaus and his colleagues showed that 7-dehydrocholesterol was converted to vitamin D 3 , and subsequent work showed that this process occurred in vivo in normal skin. 7-dehydrocholesterol is not directly converted to vitamin D 3 in the skin but rather is first converted to pre-vitamin D 3 that undergoes thermal isomerization to vitamin D 3 ( Figure 65.3 ). Several other, biologically inert, side-products such as lumisterol and tachysterol are produced during the photolysis of 7-dehydrocholesterol. Vitamin D 3 has a higher affinity for the vitamin D binding protein (VDBP) than does pre-vitamin D 3 , and the binding of vitamin D 3 to VDBP following its formation in the skin facilitates the removal of the vitamin from skin. Vitamin D 2 and vitamin D 3 are converted in the liver to 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 by the hydroxyvitamin D 25-hydroxylase without significant product inhibition of the enzyme, and consequently circulating 25-hydroxyvitamin D concentrations reflect the amount of vitamin D ingested and the amount of vitamin D formed in the skin. In accord with the earlier observation showing that sunlight exposure enhanced the formation of vitamin D 3 in the skin, several groups have shown that serum 25-hydroxyvitamin D 3 concentrations are lower during and immediately after the winter months than in the summer. In the absence of dietary vitamin D supplementation, exposure to ultra-violet B radiation plays an essential role in vitamin D production.
Following conversion of 7-dehydrocholesterol to vitamin D 3 in the skin, vitamin D 3 is transported in the plasma bound to VDBP. Any vitamin D 2 ingested in the diet is also bound to VDBP following absorption in the intestine. Vitamin D 2 and vitamin D 3 are delivered to the liver for hydroxylation by the multicomponent, cytochrome P-450 containing enzyme, vitamin D 25-hydroxylase, which is present in the liver microsomes as well as in mitochondria ( Figure 65.4 ). Hepatic conversion of vitamin D 3 to 25-hydroxyvitamin D 3 is not tightly regulated due to a lack of product inhibition of the microsomal vitamin D 3 25-hydroxylase. Several cytochrome P-450s have been cloned and shown to metabolize vitamin D 3 to 25-hydroxyvitamin D 3 , including several microsomal cytochrome P-450s and one mitochondrial cytochrome P-450. 25-hydroxyvitamin D is the serum metabolite generally measured to determine vitamin D sufficiency or insufficiency in an individual. Accurate determinations of both 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 are obtained using high-performance liquid chromatography methods and mass-spectrometry based methods and such methods are preferred to those using protein binding or antibody binding assays.
25-Hydroxyvitamin D 3 is not biologically active except in large concentrations, and it must be metabolized further in the kidney to the bioactive form of vitamin D, 1α,25-dihydroxyvitamin D 3 ( Figure 65.5 ). The 25-hydroxyvitamin D 3 -1α-hydroxylase, the enzyme responsible for the conversion of 25-hydroxyvitamin D 3 to 1α,25-dihydroxyvitamin D 3 , is a multicomponent, cytochrome P-450 containing enzyme in the mitochondria of renal proximal tubular cells. Although the kidney is the primary site of 25-hydroxyvitamin D 3 -1α-hydroxylase activity, several other cell types have been shown to have 1α-hydroxylase activity. It had previously been thought that the proximal tubule epithelial cells were the only renal cells with 25-hydroxyvitamin D 3 1α-hydroxylase activity. However, several investigators have clearly shown that this enzyme is present and active in several segments of the renal tubule. The key factors regulating 25-hydroxyvitamin D-1α-hydroxylase production and activity are depicted in Table 65.2 . Parathyroid hormone is a potent stimulator of 25-hydroxyvitmain D-1α-hydroxylase and is discussed in detail in the subsequent sections. In addition to parathyroid hormone, low serum calcium, and low serum phosphorus also stimulate 25-hydroxyvitamin-1α-hydroxylase activity. 1α,25-dihydroxyvitamin D 3 provides a negative feedback through the vitamin D receptor (VDR). VDR-knockout mice have very high concentrations of 1α,25-dihydroxyvitamin D 3 .
| Factor | Animals | Humans | References |
|---|---|---|---|
| Parathyroid hormone | ↑ | ↑ | |
| Serum inorganic phosphorus | ↓ | ↓ | |
| 1α,25(OH) 2 D 3 | ↓ | ↓ | |
| Calcium (direct) | ? | ↓ | |
| Calcitonin | ↑,↓,0 | ↑ | |
| Hydrogen ion | ↓ | 0 | |
| Sex steroids | ↑ | ↑ | |
| Prolactin | ↑ | 0 | |
| Growth hormone and insulin-like growth factor-1 | ↑ | ↑,↓,0 | |
| Glucocorticoids | ↓,0 | ↑,↓,0 | |
| Thyroid hormone | ? | ↓ a | |
| Fibroblast growth factor 23 | ↓ | ? | |
| Frizzled related protein 4 | ↓ | ? | |
| Pregnancy | ↑ | ↑ a |
a Effects may be secondary to changes in calcium, phosphorus or parathyroid hormone. ↑, Stimulation or increase; ↓, suppression or decrease; 0, no effect; ?, unknown effect.
Recently, other proteins referred to as “phosphatonins” that induce renal phosphate loss have been shown to inhibit renal 25-hydroxyvitamin-1α-hydroxylase activity and 1α,25-hydroxyvitamin D 3 production. Two such proteins include fibroblast growth factor 23 (FGF23) and secreted frizzled related protein 4 (sFRP4). Both of these proteins are able to inhibit renal tubule phosphate reabsorption which leads to hypophosphatemia. Despite the hypophosphatemia, which is a potent stimulator of 25-hydroxyvitamin-1α-hydroxylase activity, FGF23 and sFRP4 are capable of preventing the conversion of 25-hydroxyvitamin D 3 to 1α,25-dihydroxyvitamin D 3 . FGF23 and sFRP4 are over-expressed in tumors that cause oncogenic osteomalacia, a condition characterized by hypophosphatemia, hyperphosphaturia, and inappropriately low serum 1α,25-dihydroxyvitamin D 3 concentrations. These biochemical abnormalities, including the low 1α,25-dihydroxyvitamin D 3 levels, completely resolve after removal of the offending tumor. These proteins appear to play an important role in mineral and vitamin D metabolism.
Mechanism of Action of Vitamin D in Intestine
Calcium is absorbed by the intestine (predominantly in the duodenum and proximal small intestine) by two mechanisms, a passive paracellular mechanism and an active transcellular one. The movement of calcium across the apical border of the intestinal cell into the cell is down a concentration gradient (the interior of the intestinal cell has a calcium concentration in the high nanomolar range) and an electrical gradient (the interior of the cell is electronegative relative to the lumen). It does not require the expenditure of energy. However, the movement of calcium out of the intestinal cell at the basolateral membrane is against an electrical and concentration gradient and requires the expenditure of energy ( Figure 65.6 ). Essential to the process of active calcium transport are several vitamin D dependent proteins, each with a specific function. Vitamin D-dependent calcium transport proteins found in the intestinal epithelial cells include the epithelial calcium channel (ECaC), calbindin D 9K , and the plasma membrane calcium pump (PMCA). These proteins work in concert to transport calcium from the intestinal lumen, through the brush border membrane, across the intracellular space to the plasma membrane surface and finally out of the cell into the extra-cellular fluid and plasma ( Figure 65.7 ).
The actions of vitamin D in the intestine play an integral role in the maintenance of plasma calcium concentrations within a narrow range in the face of wide fluctuations in dietary calcium intake. When there is an abundance of dietary calcium, a substantial portion can be absorbed passively through the paracellular route. However, when dietary calcium intake declines and plasma calcium concentrations fall, several adaptive responses occur including the production and release of PTH from the parathyroid gland ( Figure 65.8 ). The parathyroid gland is able to sense and respond to slight changes in extracellular calcium concentrations through the calcium sensing receptor (CaSR). In addition to the direct effects on calcium homeostasis in bone and the kidney (discussed below), PTH enhances intestinal calcium absorption indirectly by increasing 1α,25-dihydroxyvitamin D 3 production in the proximal tubule cells of the kidney. Bland et al. have also demonstrated that, in vitro, 25-hydroxyvitamin D-1α-hydroxylase activity is regulated by extracellular calcium concentrations independent of PTH. They showed that human proximal tubule cells cultured in a low calcium environment resulted in a significant increase in 1α-hydroxylase activity and this activity was significantly reduced when cultured in a high calcium environment.
The response in the intestine to low dietary calcium intake is to increase the proportion of ingested calcium that is subsequently absorbed. This is accomplished by increasing the synthesis and activity of several vitamin D responsive calcium transport proteins in the intestinal epithelial cell. The first step of calcium entry from the intestinal lumen to the interior of the cell is accomplished by the ECaC located on the apical surface of the intestinal brush border epithelium. When present in the apical cell wall, ECaC facilitates the entry of calcium ions into the intracellular space. Two forms of ECaC (ECaC 1 and ECaC 2 ) are found in the intestine and both are vitamin D responsive. Vitamin D enhances ECaC mRNA expression through the vitamin D receptor (VDR). Bouillon et al. has demonstrated in VDR-KO mice that the VDR is necessary for effective regulation of several proteins involved in intestinal calcium transport including ECaC. Van Abel et al. have shown that 25-hydroxyvitamin D 3 1α,25-hydroxylase-KO mice also have lower expression of ECaC than wild-type mice which was corrected with the administration of 1α,25-dihydroxyvitmain D 3 . Although the stimulatory effect of vitamin D on calcium transport protein production in the intestine is clear, it is unclear what the direct effect of calcium may be on ECaC expression. Regardless of the difference in response to a high calcium diet between the VDR-KO and 25-hydroxyvitamin D 3 1α-hydroxylase-KO mice, these studies would suggest that ECaC expression is regulated by vitamin D-dependent and vitamin D-independent mechanisms.
After calcium ions have entered the enterocyte, they must be transported across the cell to the basolateral membrane, where they will subsequently be transported out of the cell. This is accomplished by the calbindins which are found both in intestinal epithelial cells and in renal tubule cells. In humans, calbindin D 9K is responsible for transcellular transport in the intestine and calbindin D 28K in the renal epithelium. Human calbindin D 9K is a 79 amino acid protein encoded on the X chromosome. Hisham at al. identified a putative vitamin D response element in the 5’-flanking region of the calbindin D 9K gene in the rat. It has clearly been shown that calbindin D 9K mRNA and protein expression is diminished in the face of vitamin D deficiency and that 1α,25-dihydroxyvitamin D 3 administration will increase this expression in various animal models of vitamin D deficiency. Basal expression and response to 1α,25-dihydroxyvitamin D 3 treatment changes with age in both rats and humans. Basal expression is lower and the increase in calbindin D 9K in response to a given amount of 1α,25-dihydroxyvitamin D 3 is diminished. Although 1α,25-dihydroxyvitamin D 3 appears to be the major stimulus for calbindin D 9K in the intestine, dietary calcium may have a direct effect on its expression.
The increased expression of ECaC and calbindin D 9K likely enhance the capacity of the enterocyte to transport calcium from the intestinal lumen to the intracellular space and subsequently to the basolateral membrane. However, their ability to increase calcium absorption in the face of low dietary calcium intake is dependent on the plasma membrane calcium pump (PMCA). The PMCA represents the final step in active calcium transport across the intestinal mucosa and is the single vitamin D responsive protein in the intestine that is energy dependent. Because it is energy dependent, the PMCA is able to transport calcium across the basolateral membrane against a concentration and electrical gradient. Without the PMCA, calcium absorption through the intestinal epithelial cell could not take place in a state of low calcium intake and low intestinal lumen calcium concentrations. Several isoforms of the protein have been identified with PMCA-1 being the major form found in the human duodenum. The PMCA is found in the basolateral membrane and actively transports calcium ions out of the cell into the interstitial space against a concentration gradient. Several investigators have shown that PMCA expression is decreased in the absence of, or increased by, the addition of 1α,25-dihydroxyvitamin D 3 .
Vitamin D plays an integral role in the absorption of calcium across the intestinal mucosa. Several key proteins involved in this process are directly regulated by 1α,25-dihydroxyvitamin D 3 as demonstrated by numerous experimental conditions and in many species. VDR-KO and 25-hydroxyvitamin D 1α-hydroxylase-KO mice have proven to be useful animal models in elucidating the role of vitamin D in intestinal calcium transport. In both of these models, significantly lower concentrations of all three transport proteins when compared to their wild-type counterparts have been described. In addition, intestinal calcium transport is lower in these KO models, demonstrating their importance in calcium transport. Lending further evidence to the importance of 1α,25-dihydroxyvitamin D 3 in intestinal calcium transport is the excellent correlation between increased ECaC, calbindin D 9K , and PMCA expression with improved calcium absorption in vitamin D deficient animals when 1α,25-dihydroxyvitamin D 3 is replaced. This is an effect that is apparent in young as well as older animals. However, the effects of 1α,25-dihydroxyvitamin D 3 on transport protein expression are blunted with age. This may at least partly explain the age-related decline in intestinal calcium absorption.
Mechanism of Action of Vitamin D in Kidney
Calcium handling in the kidney is similar to that in the intestine. There are both vitamin D-dependent and vitamin D-independent mechanisms at work. Reabsorption can take place through a passive paracellular route or an active vitamin D dependent transcellular route. About 55% of plasma calcium is ultra-filterable with about 98% being reabsorbed in the tubule. The kidney plays a unique role in the hormonal regulation of calcium in that it is the primary site of conversion of 25-hydroxyvitamin D 3 to the active form of the hormone 1α,25-dihydroxyvitamin D 3 . This was first discovered by Fraser and Kodicek when 1α,25-dihydroxyvitamin D 3 production was noted to be diminished after nephrectomy. Although the kidney is the major site of 25-hydroxyvitamin D 3 1α-hydroxylase activity, several other cell types are capable of converting 25-hydroxyvitamin D 3 to 1α,25-dihydroxyvitamin D 3 . It has been known for some time that the proximal tubule epithelial cell exhibited 25-hydroxyvitamin D 3 1α-hydroxylase activity. However, the presence of 25-hydroxyvitamin D-1α-hydroxylase mRNA and protein has more recently been described by Zehnder et al. in more distal portions of the tubule including the distal convoluted tubule, cortical collecting duct, thick ascending limb of loop of Henle, and Bowman’s capsule. The significance of 25-hydroxyvitamin D-1α-hydroxylase expression and activity in these distal portions of the nephron is not completely known. One could speculate there is a paracrine effect of vitamin D in these portions of the tubule since vitamin D-dependent calcium transport takes place in the distal nephron.
As previously mentioned, a state of hypocalcemia will lead to increased 1α,25-dihydroxyvitamin D 3 production in the kidney that is stimulated by higher PTH concentrations and probably by the hypocalcemia itself. Several vitamin D responsive proteins that are essentially analogous to those described in the intestine are present in the renal tubule cells. The up-regulation of these proteins by vitamin D helps defend against hypocalcemia and the maintenance of plasma calcium within the normal range in the face of low calcium intake or increased bone or soft tissue calcium deposition.
The first step in renal calcium transcellular transport involves the epithelial calcium channel. ECaCs have been described by Hoenderop et al. in the renal tubule apical membrane and are felt to be responsible for uptake of luminal calcium ions to the intracellular compartment. Of the two forms of ECaCs, ECaC-1 expression appears to be limited to the kidney whereas ECaC-2 is found in several other tissues. Human ECaC-1 is encoded on the long arm of chromosome 7 and has several putative vitamin D response elements. ECaC mRNA and protein is up-regulated in vitamin D deficient animals after supplementation with 1α,25-dihydroxyvitamin D 3 . When measured by quantitative PCR in the VDR-KO mouse model, ECaC-1 concentrations are decreased in the kidney when compared to wild-type littermates. However, in the same study, Weber et al. demonstrated that ECaC-1 could be increased by giving the animals a high calcium rescue diet. This would suggest that renal ECaC-1 is regulated by vitamin D dependent and vitamin D independent mechanisms.
Extensive work has been done regarding the role and regulation of the calcium binding protein calbindin D 28K in the kidney. It is felt that calbindin D 28K acts as an intracellular calcium transporter from the apical membrane to the basolateral membrane similar to the action of calbindin D 9K in the intestine. Calbindin D 28K is expressed not only in the kidney but also in neural tissue. However, only renal concentrations seem to be impacted by exposure to 1α,25-dihydroxyvitamin D 3 . Within the renal tubule, calbindin D 28K is found mainly in the distal portions of the nephron and co-localizes with other vitamin D responsive proteins involved in transepithelial calcium transport. In contrast to the ECaC and PMCA which are located in the apical and basolateral membranes, respectively, calbindin D 28K is found in the cytosolic compartment. Several groups have clearly shown that renal calbindin D 28K is regulated by vitamin D. Vitamin D deficient or replete animals given 1α,25-dihydroxyvitamin D 3 significantly increase the expression of calbindin D 28K mRNA and protein. This effect is also seen in vitro when cultured renal tubule epithelial cells are exposed to 1α,25-dihydroxyvitamin D 3 . Although 1α,25-dihydroxyvitamin D 3 is a potent stimulator of renal calbindin expression, changes in calcium, phosphorus, and magnesium also impact its expression. Huang and Christakos showed a lack of effect of a high calcium diet in vivo in vitamin D deficient animals on mRNA levels of calbindin D 28K . However, Clemens et al. showed that 1α,25-dihydroxyvitamin D 3 induction of calbindin D 28K was enhanced by exposure of the cells to a higher calcium concentration. Renal calbindin production also appears to be increased when exposed to a low phosphorus diet and decreased when exposed to a diet rich in magnesium.
When plasma calcium concentrations are low, the filtered amount of calcium in the glomerulus is also diminished, and the kidney must increase the proportion of calcium that is reabsorbed in order to maintain normal calcium balance. This process cannot take place without the ATP-dependent PMCA. Multiple forms of the PMCA have been described with ubiquitous distribution. As in the intestine, the renal PMCA localizes to the basolateral plasma membrane. Along with the sodium-calcium exchanger (NCX), the PMCA is responsible for transporting calcium ions out of the renal epithelial cell into the interstitial space and eventually into the blood. The NCX itself cannot transport calcium across a concentration or electrical gradient as can the PMCA. Renal PMCA mRNA and protein are up-regulated by exposure to 1α,25-dihydroxyvitamin D 3 in vivo and in vitro . Hoenderop et al. performed an experiment comparing homozygous 25-hydroxyvitamin D 3 -lα-hydroxylase knock-out mice with heterozygotes (controls). When on a normal calcium diet, homozygous mice had lower levels of ECaC-1, calbindin D 28K , and NCX concentrations when measured by real time PCR. However, PMCA concentrations were not different between the two groups. When given 1α,25-dihydroxyvitamin D 3 , both the homo- and heterozygous mice increased expression of all of the calcium transporting proteins including PMCA. This would suggest that 1α,25-dihydroxyvitamin D 3 may not be necessary for basal mRNA levels to be present but the protein is highly responsive to 1α,25-dihydroxyvitamin D 3 .
Parathyroid Hormone
Endocrine System
Parathyroid hormone is an 84 amino acid peptide produced and secreted by the parathyroid glands and is an important regulator of extracellular calcium concentrations. PTH secretion from the parathyroid gland is stimulated by hypocalcaemia and inhibited when exposed to elevated calcium levels. In turn, PTH regulates serum calcium concentrations through direct mechanisms in the kidney (enhanced calcium reabsorption) as well as in bone (release of mineral from bone). Since PTH is a potent stimulator of 1α-hydroxylase activity resulting in higher 1α,25-dihydroxyvitamin D 3 concentrations, it also exerts its effects on calcium metabolism indirectly through the vitamin D endocrine system described above. The following section will outline the regulation of PTH synthesis and secretion as well as its effects on calcium homeostasis in the kidney and in bone.
Regulation of Parathyroid Hormone
Synthesis and Secretion
The chief cells of the parathyroid gland are responsible for the synthesis, storage, and release of parathyroid hormone. PTH is the product of a larger molecule, pre-pro-parathyroid hormone. The pre-sequence is cleaved in the endoplasmic reticulum resulting in pro-PTH. The pro-sequence is subsequently removed in the Golgi apparatus, leaving the final 84 residues that constitute the secreted and active form of PTH. The release of preformed PTH from secretory vesicles is most acutely affected by extracellular calcium concentrations. Figure 65.9 depicts the steep inverse sigmoidal relationship between calcium concentrations and PTH release. A minimal increase in extracellular ionized calcium will inhibit PTH release. Likewise, a slight decrease in plasma ionized calcium is a potent stimulus for PTH synthesis and secretion.
The parathyroid cell is able to detect these small changes in calcium concentrations by means of the calcium sensing receptor (CaSR) located on the cell surface. The CaSR is not only expressed on the surface of parathyroid cells but also in the kidney, bone, intestine, and several other tissues. Mutations in the CaSR have contributed to our understanding of its importance in normal calcium homeostasis. In humans, both activating and inactivating mutation is the CaSR have been described. Inactivating mutations result in the condition Familial Hypocalciuric Hypercalcemia (FHH) in which serum calcium levels are increased as a result of a higher calcium set point required to inhibit PTH secretion. Similar clinical features have been described in patients with acquired disease due to inactivating antibodies against the CaSR. In both of these diseases, the CaSR has become less sensitive to the effects of extracellular calcium and thus higher concentrations are required to inhibit PTH secretion. The opposite scenario has also been described in which activating mutations in the CaSR or antibodies against the CaSR result in inherited or acquired hypoparathyroidism, respectively. These are conditions in which the CaSR is more sensitive to extracellular ionized calcium concentrations and PTH secretion is inhibited at lower calcium levels.
Several investigators have also shown that cells from parathyroid adenomas and hyperplastic parathyroid glands in patients with primary and secondary hyperparathyroidism have fewer detectable calcium sensing receptors than normal parathyroid tissue. These clinical scenarios point to the critical role of the CaSR in the regulation of PTH secretion in response to calcium concentrations in humans.
Parathyroid hormone secretion is also regulated by 1α,25-dihydroxyvitamin D 3 through the VDR. The response to vitamin D on PTH secretion is not as rapid as the acute changes seen with fluctuations in extracellular calcium concentrations detected by the CaSR. Vitamin D acts as a negative feedback on PTH secretion as a means to prevent hypercalcemia. When faced with hypocalcemia, preformed PTH is rapidly secreted from the parathyroid cell and acts in the kidney to increase 1α-hydroxylase activity. The result is increased conversion of 25-hydroxyvitamin D 3 to 1α,25-dihydroxyvitamin D 3 . Vitamin D response elements (VDRE) have been identified in the 5’ flanking region of the parathyroid gene and serve to inhibit PTH gene transcription resulting in decreased hormone synthesis. In addition to inhibiting PTH secretion by means of the CaSR, extracellular calcium concentrations also influence PTH secretion indirectly by altering VDR expression. Brown et al . fed three groups of rats a vitamin D deficient diet along with either a low, normal, or high calcium diet. Parathyroid tissue VDR mRNA concentrations were approximately sixfold higher in the vitamin D deficient rats fed a high calcium diet. Up-regulation of the VDR in parathyroid cells by higher extracellular calcium concentrations has been observed by others, both in vivo and in vitro , showing the integrated control of PTH secretion by calcium ion concentration and vitamin D. These findings would suggest that the negative feedback of vitamin D on the parathyroid gland is augmented by higher calcium concentrations, adding yet another layer of control in the regulation of normal calcium homeostasis.
Several other factors also play a role in PTH secretion including serum phosphorus concentrations, magnesium and certain amino acids.
Parathyroid Hormone and Kidney
The parathyroid hormone receptor (PTHr) is a G-protein coupled receptor found in a diverse array of tissues including the kidney and bone where it plays an integral part in calcium homeostasis. Using RT-PCR and in-situ hybridization techniques, the PTHr has been identified in several portions of the nephron including the glomeruli, proximal convoluted tubule, proximal straight tubule, medullary and cortical thick ascending limb, and the distal convoluted tubule. PTH serves different functions in mineral metabolism along the nephron including decreasing phosphate reabsorption (primarily in the proximal portion), facilitating calcium reabsorption (distal portion), and enhanced conversion of 25-hydroxyvitamin D 3 to 1α,25-dihydroxyvitamin D 3 .
The direct effect of PTH on transcellular reabsorption of filtered calcium takes place primarily in the distal nephron and uses some of the same machinery as vitamin D regulated calcium transport. Calcium entry into the renal tubule epithelial cell through calcium channels is enhanced in the presence of PTH. Friedman et al. demonstrated that this process is dependent on both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and calcium transport could be prevented with specific inhibitors of PKA or PKC.
Using a murine distal convoluted tubule cell line, Gesek and Friedman demonstrated a dose-dependent calcium uptake by PTH that was inhibited by a dihydropyridine calcium channel blocker. They also demonstrated in the same study that hyperpolarization of the cell was responsible for the calcium uptake and the hyperpolarization was due to chloride efflux. They proposed that the principal mechanism by which PTH induced calcium reabsorption in the distal tubule was through activation of chloride channels that resulted in epithelial cell hyperpolarization. This hyperpolarization sets up a favorable gradient for transcellular calcium transport, thereby facilitating its reabsorption from the distal tubule.
In addition, it has also been shown that distal tubule cells exposed to PTH have increased sodium dependent calcium transport. These findings demonstrate the synergistic effects of PTH and vitamin D on calcium reabsorption in the kidney. As outlined in the sections above, there are several vitamin D dependent calcium transport proteins in the distal portions of the nephron. Several of these proteins are utilized or their activity is up-regulated in the presence of PTH.
PTH not only has direct effects on renal tubular calcium transport but also exerts indirect effects through the vitamin D system. PTH is a potent stimulator of 25-hydroxyvitamin D 3 1α-hydroxylase activity in the kidney leading to higher 1α,25-dihydroxyvitamin D 3 concentrations. Using a luciferase reporter, Brezna et al. demonstrated that PTH stimulates 1α,25-dihydroxyvitamin D 3 production through a promoter in the 1α-hydroxylase gene. Vitamin D then serves to increase extracellular calcium concentrations by the mechanisms describe above.
In states of renal dysfunction, the effects of PTH on the kidney can be dramatically reduced. Its ability to promote phosphate loss in the proximal tubule and reabsorb calcium in the distal tubule can be diminished despite high circulating levels of PTH. The up-regulatory effect of PTH on 25-hydroxyvitamin D 3 1α-hydroxylase activity is significantly reduced in renal failure. These findings in renal failure are probably (at least partially) due to the decreased number of PTHr found in renal tubular cells in humans and in animal models of chronic renal insufficiency.
Parathyroid Hormone and Bone
Calcium metabolism in bone regulated by PTH is complex and involves both osteoblasts and osteoclasts. The effects of PTH on osteoclasts had been thought to be primarily indirect since they do not normally contain PTH receptors. However, osteoclasts from patients with secondary hyperparathyroidism do express PTHr mRNA and protein suggesting a possible direct effect of PTH on ostoeclasts in the disease state. In addition, Dempster et al. have demonstrated increased bone resorption by normal human osteoclasts derived from peripheral monocytes grown on bone slices when exposed to PTH. This effect was seen in the absence of osteoblasts.
When discussing the role of PTH on calcium homeostasis, the main effect in bone is to release calcium ion into the extracellular compartment to maintain normal ionized calcium concentrations. Osteoclasts are the cells responsible for bone resorption and release of mineral. The stimulation of osteoclastogenesis by osteoblasts is by way of the receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), RANK system ( Figure 65.10 ). RANKL production by osteoblasts is stimulated by PTH and OPG is inhibited by PTH. RANKL induces osteoclast differentiation and activation through the RANK receptor. OPG acts by binding to RANKL thus inhibiting osteoclast activation through the RANK receptor. Since PTH has positive and negative effects on the production of RANKL and OPG, respectively, its effects on resorption are through two separate yet integrated mechanisms. Once osteoclasts have been activated by PTH through this indirect mechanism, bone resorption ensues and calcium is released.
