Calcium and phosphate homeostasis involves interrelated actions of parathyroid hormone (PTH), vitamin D ( the active form of vitamin D is 1,25(OH)2D (calcitriol), and calcitonin. These hormones influence calcium and phosphate release from bones (osteolysis) and excretion or resorption from the kidneys or intestines. Phosphatonins are recently discovered regulators of phosphate homeostasis (Cavalli et al., 2012). Other hormones that can influence calcium and phosphate to a lesser extent or under specific conditions are: estrogens, glucagon, prolactin (lactation only), corticosteroids and growth hormone. Pathologic changes in these other hormones can also result in changes in calcium and phosphate levels.
Parathyroid hormone (PTH) is secreted by chief cells of the parathyroid gland. The net effect of PTH is increased calcium and decreased phosphate in serum. Production of PTH is controlled mostly by free ionized calcium levels, which are sensed by specific calcium receptors (G-protein coupled receptors) on chief cells. Activation of the receptor (in response to high free ionized calcium) inhibits PTH secretion, whereas inhibition of the receptor (e.g. low free ionized calcium) stimulates PTH secretion. PTH has a very short half life (3-5 minutes) and must be secreted continuously by the chief cells to maintain serum levels.
- Stimulation of secretion: Decreased free ionized calcium is the main stimulus for secretion, but dopamine, prostaglandin-E2 (PGE2) and epinephrine, along with neural transmitters, can have mild effects on PTH secretion. Acidemia also stimulates PTH secretion, independently of free ionized calcium values (Lopez et al., 2003).
- Inhibition of secretion: Increased free ionized calcium (causes intracellular degradation of PTH), increased vitamin D (inhibits PTH mRNA synthesis and stimulates expression of the calcium-sensing receptor which inhibits PTH secretion – this usually only occurs when free ionized calcium is normal). Both high and low free ionized magnesium concentrations may inhibit PTH secretion (free ionized magnesium can be an agonist for the calcium-sensing receptor which inhibits PTH secretion; mechanism is unclear for low magnesium concentrations). Alkalemia inhibits PTH (independently of free ionized calcium values) and blunts the PTH response to hypocalcemia (Lopez et al 2003).
Parathyroid hormone has two primary sites of action: the kidney and bone.
- Kidney: Parathyroid hormone enhances renal resorption of calcium in the distal tubules, collecting ducts and ascending limb of the loop of Henle, and promotes excretion of phosphate in the proximal renal tubules. It also activates mitochondrial α1-hydroxylase in the kidney, which converts vitamin D to its active form, 1,25(OH)2D (calcitriol).
- Bone: PTH acts primarily via stimulating osteoblasts to secrete factors which promote osteoclasts to develop from monocytes, specifically RANKL (Receptor Activator of Nuclear Kappa B Ligand). RANKL binds to its receptor (RANK) on osteoclasts triggering differentiation (with monocyte-colony stimulating factor or M-CSF), preventing osteoclast apoptosis and promoting bone resorption by the release of peptidases which lyse bone matrix (osteolysis); the end result being the release of calcium (and phosphate) from bone stores. Although PTH acts to release phosphate from bone through osteolysis, its phosphaturic actions (promoting phosphate excretion in urine) dominates, so the net effect of high PTH is decreased phosphate.
Parathyroid hormone can be measured by immunoassay. Assays for detection of the intact molecule (iPTH) or amino-terminal end should be used, as assays for detection of the carboxy-terminal end of PTH are falsely increased with decreased GFR. Interpretation of PTH levels should be done concurrently with knowledge of free ionized calcium values, e.g. a normal PTH concentration in an animal with a high free ionized calcium is inappropriate and compatible with a diagnosis of primary hyperparathyroidism.
Primary hyperparathyroidism genetic testing is available from the Molecular Diagnostic section at the Animal Health Diagnostic Center. The preferred sample is EDTA plasma (separated from cells) or serum (collected into a red top and separated from cells). Samples should be frozen (and stay that way during transit) because proteases degrade the hormone.
Measurement of intact parathyroid hormone (iPTH), free ionized calcium (iCa2+), and 25-hydroxyvitamin D (calcidiol) can help discriminate between the various causes of hypercalcemia in dogs. Guidelines for interpretation of these tests in combination are shown in the table at right. Note, that parathyroid hormone-related protein may be increased in humoral hypercalcemia of malignancy if the tumor secretes this hormone.
|Primary hyperPTH||Normal or High||High||–|
|Vitamin D intoxication||Low||High||High|
Parathyroid hormone-related protein
Parathyroid hormone-related protein (PTHrP) is produced by several different cell types including lymphocytes, squamous epithelium, endocrine glands, bone, skeletal and smooth muscle and the kidney. The precise role of the protein is not known, but it is thought to be important for movement of calcium across membranes in the neonate and in the mammary gland. It is not involved in calcium homeostasis in physiologic states. PTHrP has a similar amino-terminal end to PTH and binds to PTH receptors. Therefore, PTHrP has a similar effect on serum calcium (increased total and free ionized) and phosphate (decreased) as PTH. PTHrP is secreted by apocrine anal sac adenocarcinomas, some lymphomas and squamous cell carcinomas. It is thought to be the main hormone for the paraneoplastic hypercalcemia seen in these disorders (humoral hypercalcemia of malignancy). PTHrP can also be increased in some dogs with granulomatous inflammation and may be the cause of hypercalcemia in these dogs. Specific assays for PTHrP are available in dogs and cats.
Vitamin D is ingested as vitamin D3 (or vitamin D2 in plants). Vitamin D3 (cholicalciferol) is also produced in the skin from cholesterol under the influence of UV light. When produced in the skin or ingested, vitamin D3 is transported via vitamin D-binding proteins to the liver where it is converted (by the enzyme 25 hydroxylase) to calcidiol (25HCC (hydroxycholicalciferol) or 25(OH)D). This also binds to vitamin D binding protein and is the form of vitamin D usually measured in the laboratory. Calcidiol is then filtered through the urine, where it is taken up (along with vitamin D binding protein) by specific receptors on proximal renal tubule cells. Within the tubular cells, vitamin D is converted to its most active form, calcitriol (1,25DHC (dihydroxycholicalciferol) or 1,25(OH)2D) by the enzyme, α1-hydroxylase in the proximal renal tubules (this enzyme is absent in horse kidneys). Since skin production of vitamin D3 is ineffective in most domestic animals (due to the hair coat, which prevents UV light from acting on skin cells and the presence of a reductase enzyme in skin in dogs and cats), animals typically rely on dietary absorption of vitamin D to maintain vitamin D levels (Parker et al 2017). Liver production of vitamin D is not regulated and is primarily dependent on the amount of vitamin D absorbed from the diet in animals. The different forms of vitamin D are summarized below.
|Vitamin D3||Cholecalciferol||Ingested or produced in skin from cholesterol under the influence of UV light. Least active form. Transported to liver with vitamin D-binding proteins.|
|25HCC||Calcidiol or 25 hydroxycholecalciferol or 25(OH)D||Produced in the liver from cholecalciferol by 25-hydroxylase, filtered in urine and re-absorbed by proximal renal tubules via specific receptors (e.g. cubilin). More active than cholecalciferol (up to 5x). Usually measured.|
|1,25DHC||Calcitriol or 1,25 dihydroxycholecalciferol or 1,25(OH)2D||Converted from 25HCC in proximal renal tubules (absorbed from urine) by (facilitated by PTH). Most active form of vitamin D (25x more than calcidiol). Infrequently measured.|
|24,25 DHC||Hydroxycalcidiol or 24,25 dihydroxycholecalciferol or 24,25(OH)2D||Converted from 25HCC in the liver by a cytochrome P450 hydroxylase enzyme. It is a less active form of vitamin D (like cholecalciferol) and is excreted in urine and bile. This form of vitamin D is present in the horse (as is 25 HCC and vitamin D3).|
The enzyme α1-hydroxylase is present in other cells, e.g. macrophages, and calcitriol is responsible for the hypercalcemia of malignancy seen in some dogs with lymphoma and possibly histiocytic sarcoma. Excess production of vitamin D by macrophages is also thought to be the main mechanism behind hypercalcemia in granulomatous disease, e.g. fungal diseases and Angiostrongylus vasorum infection in dogs, idiopathic granulomatous disease in horses.
Formation of calcitriol is primarily influenced by free ionized calcium levels, but also by PTH and phosphate.
- Stimuli: PTH, decreased free ionized calcium, decreased phosphate.
- Inhibitors: Calcitriol (self negative feedback – it inhibits α1-hydroxylase), FGF23, increased free ionized calcium, increased phosphate. Note, that horses lack 1α-hydroxylase and thus cannot convert vitamin D to its most active form. They likely do not need as much vitamin D as other species because they have a high content of calcium in their diet. They do, however, have a less active form of vitamin D (24,25 dihydroxycholecalciferol), which can promote intestinal resorption of calcium and phosphate.
Calcitriol acts on the intestines primarily and kidney.
- Intestines: Calcitriol increases calcium and phosphate resorption, which may lead to high total calcium and phosphate in serum. Vitamin D induces the production of calbindins (a family of calcium-binding proteins), particularly calbindin-D9k, in the intestinal mucosa. Calbindin-D9k facilitates the transport of calcium across the luminal surface of the intestinal epithelial cells through calcium channels and may also increase the activity of the basolateral pump that transfers calcium into blood from the intestinal cell.
- Kidney: In the kidney, calcitriol promotes calcium resorption in the distal convoluted tubule by increasing levels of calbindin (a family of calcium binding proteins).
- Bone: Calcitriol facilitates the action of PTH on osteoblasts (promoting osteolysis and increased calcium and phosphate) but can also stimulate bone resorption by stimulating RANKL production from osteoblasts or stromal cells. Vitamin D can also inhibit PTH secretion by downregulating gene synthesis (this only occurs when free ionized calcium is normal).
Calcitonin is produced by parafollicular cells (C cells) in the thyroid gland. Calcitonin acts on the bone and kidney and counters the calcium- raising effects of vitamin D and PTH by inhibiting osteolysis and stimulation of renal excretion of calcium and phosphate. Its main physiologic response is to limit the post-prandial rise in calcium and it acts (albeit ineffectively) as an emergency response to free ionized hypercalcemia. It can be measured in dogs using radioimmunoassay, but this is infrequently done.
- Stimuli: Increased free ionized calcium, increased free ionized magnesium, β-adrenergic stimulation, dopamine, estrogen, gastrin, cholecystokinin, and glucagon.
- Inhibitors: Decreased free ionized calcium, somatostatin.
These are hormones that regulate phosphate balance. There are several of these, including fibroblast growth factor-23 (FGF-23), sFRP-4 and MEPE. Of these, FGF-23 seems to be a key hormone. FGF-23 is produced in osteocytes and osteoblasts. It acts on the proximal renal tubular epithelial cells (which usually absorb most of the filtered phosphate) by binding to receptors on cells, that stimulate signaling, resulting in inhibition of phosphate resorption. This is accomplished by downregulation of the luminal sodium/phosphate cotransporter. FGF-23 also reduces synthesis of 1α-hydroxylase, the enzyme in the kidney responsible for activation of vitamin D (calcidiol to calcitriol) and high FGF23 concentrations is thought to be one of the main mechanisms for low vitamin D in chronic kidney disease (Hardcastle and Dittmer 2015). Phosphatonins can also inhibit PTH. Thus, phosphatonins promote phosphate excretion and decrease intestinal phosphate absorption (less active vitamin D). If in excess, FGF-23 can result in hypophosphatemia. Conversely, if phosphatonins are deficient or dysfunctional (due to the Kloth receptor defects), hyperphosphatemia can result (from decreased renal excretion of phosphate). The Kloth receptors are abnormal in renal disease which promotes hyperphosphatemia and PTH secretion despite higher levels of FGF-23 (Hardcastle and Dittmer 2015).
- Stimuli for FGF-23 synthesis and secretion are calcitriol, increased serum phosphate (Cavalli et al., 2012) and decreased GFR (mechanism unclear [Hardcastle and Dittmer 2015]). FGF-23 is also produced in soft tissue sarcomas in dogs (Hardcastle and Dittmer 2015).
|Overall function||Regulation of the calcium/phosphate ratio||Maintain minerals for bone formation||Limit postprandial rise in calcium, emergency control of hypercalcemia||Regulate phosphate excretion|
|Source||Chief cells – parathyroid gland||Kidney – this organ performs the final activation (1α-hydroxylation) step (except in horses)||C-cells (parafollicular cells) of thyroid gland||Osteoblast, osteocyte|
|Control of release||Stimulate: ↓ iCa2+ (free ionized calcium), ↓ calcitriol
Inhibit: ↑ iCa2+, ↑ calcitriol
|Stimulate: ↓ iCa2+, ↓ PO43-, ↑ PTH
Inhibit: ↑ iCa2+, ↑ PO43-, ↑ calictriol
Low: ↓ renal mass (not in horses)
|Stimulate: ↑ iCa2+, Mg2+, gastrin, cholecystokinin, glucagon.
Inhibit: ↓ iCa2+, ↑ somatostatin
|Stimulate: Calcitriol (active vitamin D), ↑ PO43-|
|Target organs||Bone: Mobilise calcium, phosphate
Kidney: ↑ calcium resorption, ↑ phosphate excretion
Intestine: ↑ calcium uptake (via Vit D)
|Intestine: ↑ calcium and phosphate uptake
Kidney: ↑ calcium resorption (via PTH mostly)
Bone: Mobilize calcium, phosphate (via PTH)
|Bone: Inhibit osteoclastic bone resorption (↓ calcium)
Kidney: inhibits calcium and phosphate resorption (↓ values)
|Kidney: ↑ phosphate excretion
Intestine: ↓ phosphate uptake (↓ α1-hydroxylase and ↓ vitamin D)
Bone: Inhibit osteoclastic bone resorption (decrease phosphate) via inhibition of PTH
|Net effect||↑ calcium (free ionized and total)
↓ phosphate (phosphaturic effects dominate)
|↑ calcium (free ionized and total)
↑ phosphate (pathologic states)
|↓ calcium (free ionized and total)