S3: Control of Calcium and Phosphate Metabolism

Question 1

Functions of Calcium

Answer 1

• It is important for bone growth and remodelling (mineral element of skeleton)
• For secretion
• In muscle contraction
• Blood clotting
• As a co-enzyme
• Stabilisation of membrane potentials (in nerves and muscle is important in maintaining negative intracellular charge)
• Secondary messenger

Question 2

Name places in the body where calcium is found and in what form is it found?

Answer 2

Majority of calcium is stored in the bone (99% in skeleton). The rest is extracellular and there is a minute amount intracellular because it is an signalling molecule.

• Around 45% is ionised and free
• Around 45% is bound to plasma proteins
• A little is bound to ions e.g. phosphate, lactate, HCO3-

Question 3

What are the two primary hormones affecting calcium balance?

Answer 3

The extracellular calcium levels are controlled by PTH and vitamin D (endocrine system).

Question 4

Functions of phosphate (H2PO4- and HPO42-)

Answer 4

• It is an element in high energy compounds such ATP and also secondary messengers like cAMP
• It is a constituent of DNA/RNA, phospholipid membranes and in bone
• It is also involved in the activation of enzymes by phosphorylation

Question 5

Name places in the body where phosphate is found and in what form is it found?

Answer 5

It is mainly found in the skeleton (90%) and around 10% is intracellular with a small amount extracellular.
- Half is free and half is bound to proteins

Question 6

What hormones control phosphate balance?

Answer 6

The control of extracellular phosphate is by the kidneys and also effects of PTH and FGF23 (fibroblast growth factor 23).

Question 7

Describe the daily turnover of calcium and phosphate

Answer 7

We have a daily turnover of calcium and phosphate, we take both in via the diet and we excrete through faeces and urine.
Generally it is kept in balance, most is kept in the skeleton which acts as a buffer. So if we want to make changes to these elements in the blood, it is generally bone that will be stimulated to release the elements from it or take them back in and this low rate turnover can be regulated.

Question 8

How does PTH, vitamin D and FGF23 affect calcium and phosphate turnover?

Answer 8

• PTH and Vitamin D are important in delivering calcium and phosphate to the bone
• PTH is important in controlling the flux of calcium in the kidney
• PTH and FGF23 controls the flux of phosphate in the kidney
• Vitamin D does act on the bone but its main action is in bringing calcium and phosphate through the gut from the diet.

Question 9

Is bone static?

Answer 9

No! It is often thought of being quite static when it is in fact an active living tissue and constantly is being remodelling and reformed.

Question 10

Types of cells in bone

Answer 10

• Osteoblasts build up bone
• Osteoclasts that model and take out bone
• Osteocytes which are osteoblasts that have finished the active growth phase and are trapped in the boney maxtrix. They are quiet inert cells.

Question 11

What are the two types of bone?

Answer 11

1. Corticol bone which is more involved in strength
2. Trabecular bone which is involved in buffering area of extra stress. It is more loose and net like.
   e. g. long bones have an outside sheath of cortical bone but the trabecular bone ob the inside acts as bracing.

Question 12

Describe stages of bone remodelling

Answer 12

1. Differentiation of stem cells to osteoclasts.
2. Osteoclasts come in and carve a pit in bone by ‘reabsoring it’.
3. Macrophages come in and mop up any debris and bacteria.
4. Osteoblasts come into put and lay down new boney matrux (new osteoid and minerals) to fill the hole.
5. Some of the osteoblasts get stuck and covered by the new boney matrix and becomes osteocytes.

Question 13

Describe cellular origin of osteoblast and osteoclast

Answer 13

• The osteoclast is actually a modified macrophage, it comes from the hemapoietic stem cells.
• The osteoblast is a mesenchymal derived cell. However, if the conditions are right, some of the hematopoieic stem cells can differentiate into osteoblasts.

Question 14

Difference osteoblast and osteoclast

Answer 14

The osteoblast cells when active are osteoblasts but then they may become mononuclear cells (the osteocyte) when they become inactive after the end of the bone resorption unit.

Question 15

What activates the osteoclast for reabsorption?

Answer 15

The osteoclast precursor is activated by RANK ligand which is released by osteoblasts. The osteoclast is dervied from its
precursor being activated.
- When there is a signal for bone resorption e.g. PTH, the osteoblast comes active and releases RANK ligand and activates the RANK receptor on the osteoclast precursor and via activation of nuclear kappa beta stimulates gene transcription and differentiation into osteoclasts.
- The growth of the precursor is promoted by GM-CSF produced by T-cells locally

Question 16

What is OPG (oesteoprotegerin)/OCIF (osteoclastgenesis inhibitory factor) role?

Answer 16

OPG/OCIF bind to RANK which inhibits differentiation of osteoclasts causing bone to be laid down.

Question 17

What stimulates reabsorption and where is its receptor found?

Answer 17

PTH stimulates resorption. The PTH receptor is on the osteoblast not osteoclast, this then allows co-ordinated resorption of bone.

Question 18

How do osteoblasts know where stress is on bone?

Answer 18

Bone is laid down along the lines of stress. Osteoblasts know where stress is because some electricity is produced when you stress the bone and it tells the osteoblasts when to switch on.

Question 19

How does bone act as an endocrine organ (FGF23 and uCON)?

Answer 19

Osteocytes produce fibroblast growth factor 23, it acts on the kidney to decrease synthesis of active vitamin D and to increase excretion of inorganic phosphate (when too much in the body).
Osteoblasts produce uncarboxylated osteocalcin, this acts on pancreatic beta cells to increase insulin production and secretion, it acts on adipocytes to increase adiponectin and on muscle to increase insulin sensitivity and glucose uptake (bone also regulate metabolism to deliver energy to cells in bone for growth). Osteocalcin allows energy trapping within cells so energy can be provided for boney growth.

Question 20

What are other hormones (not FDF23 and uCON) involved in bone turnover and bone reabsorption?

Answer 20

• Sex steroids have an positive effect on bone growth by stimulating osteoblast precursors. Long term use can result in osteoporosis.
• Growth hormone also stimulates production of bone via IGF-1
• Thyroxine is needed for bony growth
• Glucocorticoids inhibit osteoblast maturation

Question 21

Hormones affecting osteoblast precursor

Answer 21

Positive:

• Eostrogen
• Androgens
• GH/IGF-1

Negative:
- Glucocorticoids

Question 22

Hormones affecting osteoclast precursor

Answer 22

Positive:

• Thyroxine
• Vitamin A

Negative:

• Eostrogen
• Calcitonin

Question 23

What cells produce PTH (parathyroid hormone)?

Answer 23

Chief cells which are very small produce PTH. They are oxyphil cells which don’t produce hormone.

Question 24

Anatomy of parathyroid gland

Answer 24

• 4
• Located on upper and lower poles of each lobe of the thyroid gland
• Supplied blood from the inferior thyroid arteries

Question 25

Embryology of parathyroid gland

Answer 25

Embryologically the parathyroid gland develops from 3rd and 4th pharyngeal pouches.

• From the dorsal part of the 3rd pharyngeal pouch, parathyroid III arises becoming the inferior parathyroid gland.
• Parathyroid IV arises from the dorsal portion of the 4th pharyngeal pouch; it separates from the pouch and migrates caudally, but ultimately becomes the superior parathyroid gland.

Question 26

Describe the synthesis of PTH

Answer 26

Parathyroid like many hormones is first made as a prepro hormone and then turned into a prohormone and then active PTH.

• The pre is first translated and it is a signal sequence. It tells the peptide to go join ER and it is cleaved off forming the prohormone.
• Once at golgi apparatus, the pro acts to prevent the peptide being broken down before it is released.
• Once the hormone is ready to be released, the pro part of the peptide is cleaved off and the active hormone is put into a vesicle and released into the circulation.

Question 27

What happens once the PTH has performed its function in the circulation?

Answer 27

After PTH has had its action in the circulation, there are degrading enzymes in the plasma which cut the PTH into various lengths.
This makes PTH biologically inactive.
- Only 20% of circulating PTH is biologically active (i.e. full length).

Question 28

Why do clinicians have problems detecting PTH and what is the solution?

Answer 28

PTH is difficult to measure because not only will we be measuring the full peptide in its active form but also measuring the inactive fragments (from degraded PTH).
The solution is to use two antibodies to detect PTH, one for the active end and another for the inactive fragment. This allows us to just measure the active one. It is called an immunometric assay.

Question 29

Why do clinicians have problems detecting calcium?

Answer 29

Calcium in the blood stream free and bound are in equilibrium. An increase in plasma proteins and alkalosis decreases ionised (free) calcium and a decrease in plasma proteins and acidosis increases ionised calcium. During acidosis, H+ binds to plasma proteins causing displacement of Ca2+ making it free in the circulation. This is important in blood tests because using a tonique can lead to acidosis leading to a false high reading of calcium so it needs to be removed to get a free blood sample.

Question 30

Describe the function and mechanism of a calcium sensing receptor

Answer 30

The ionised (free) calcium is directly sensed by a calcium receptor that sits on the PTH producing chief cell. It is a ultra-short feedback loop because the cell itself responds to the calcium around it.
When the sensor is activated, the Gaq and Gai pathway is activated so there is an increase in PLC and decrease in cAMP (via decrease in AC). IP3 goes on the increase intracellular calcium levels and there is an overall DECREASED PTH PRODUCTION.

Question 31

Described the relationship between serum calcium and PTH secretion

Answer 31

The relationship between serum Ca2+ and PTH secretion is a steep one. Once ionised calcium goes outside the normal range, PTH secretion immediately shuts off. Because Ca2+ has to be tightly regulated.
Therefore as calcium increases, PTH decreases.

Question 32

Describe PTH levels during hypercalcemia

Answer 32

If someone has hypercalcemia there should be no circulating parathyroid hormone. If PTH is high or normal, it is evidence of a problem with PTH production.

Question 33

Actions of Parathyroid Hormone

Answer 33

• It stimulates osteoblasts to produce M-CSF and RANK ligand, which increases bone reabsorption by stimulating osteoclast differentiation (takes calcium out of bone).
• It increases calcium reabsorption in the distal convoluted tubule of the kidney.
• It increases phosphate excretion.
• It increases 1-alpha hydroxylase in the PCT.
• Inhibits vitamin D and increases the breakdown of it in the kidney.
  So PTH is produced in response to low calcium in order to increase calcium.

Question 34

Describe actions of PTH in the kidney

Answer 34

• PTH stimulates AC to increase cAMP which activates PKA. PKA increases the amount of calcium channels on the luminal surface in the kidney.
• PTH also stimulates the activity of the 3Na+/Ca2+ exchanger and the Ca2+ ATPase.

Question 35

How is calcium transported from kidney lumen to interstitium

Answer 35

Calcium is taken up from the lumen of the kidney by Ca2+ channels present on the luminal membrane of the DCT.
The entry of Ca2+ into the tubular cells is driven by steep electrochemical gradient between the filtrate and cytoplasm.
Ca2+ is then bound to and transported to the basolateral membrane by calbindin.
On the basolateral side, the Ca2+ ATPase kicks calcium out as well as the 3Na+/Ca+ exchanger.

Question 36

What is PTH-related peptide?

Answer 36

In foetal life we do not produce PTH, rather we produce something called PTHrp (embryonic form of PTH produced by liver). PTHrp production stops after birth.

Question 37

Difference PTH and PTHrp

Answer 37

• PTHrp binds to the PTH1R receptors and does not stimulate 1a-hydroxylase
• PTH binds both receptors and stimulates 1a hydroxylase in the PCT

Question 38

Clinical significance of PTHrp

Answer 38

In tumours, cells are often immature and go backwards therefore we see levels of PTHrp in cancer. This increases bone reabsorption and hypercalcemia.

Question 39

Describe the synthesis of the active form of vitamin D3

Answer 39

• Vitamin D is formed when light hits the skin.
• It results in a reaction where cholesterol is reduced to cholecalciferol (vitamin D3).
• This vitamin D is made in the skin and then it passes to the liver where a hydroxyl group is added. This is unregulated and just occurs.
• The next step is regulatory and it occurs in the kidney. There is an enzyme called 1-alpha hydroxylase in the kidney and this enzyme regulates the production of active vitamin D (calcitriol).

Question 40

Sources of Vitamin D

Answer 40

• De novo synthesis of vitamin D3 where cholesterol is reduced to cholecalciferol (vitamin D3)
• Ergosterol from diet which is converted to ergocalciferol (vitamin D2)

Question 41

What happens when 1-alpha hydroxylase doesn’t work in the kidney?

Answer 41

This is associated with kidney disease where there is low levels of calcium due to low levels of vitamin D.

Question 42

Why do some granulomatous diseases e.g. sarcoidosis, tuberculosis result in hypercalcemia?

Answer 42

This is because the only other tissue type that has significant expression of 1-alpha hydroxylase is the macrophage. In granulomas where you havelarge collections of macrophages, there is enough 1-alpha hydroxylase in the granulomas to cause hypercalcaemia (due to increased active vitamin D - (calcitriol).

Question 43

Describe vitamin D receptors

Answer 43

Vitamin D acts via the vitamin D receptor which is a type 2 nuclear receptor. Vitamin D binds to the vitamin D receptor which then dimerises with the retinoic acid receptor.
This receptor then binds directly to the gene and regulates expression of the downstream genes.

Question 44

What are the two types of nuclear receptor for hormones?

Answer 44

Type 1 and type 2. In both types the hormone must be in a dimer in order to bind to the receptor that will bind directly to the gene to regulate its expression.

• In the type 1 receptor two molecules of hormone align and bind to the receptor, which then binds to the hormone response element on the gene e.g. cortisol
• In type 2 the hormone binds to the receptor which dimerises with another receptor. The receptor then binds directly to the gene and regulates expression of downstream genes. e.g. vitamin D receptor

Question 45

How does vitamin D affect PTH?

Answer 45

Vitamin D inhibits the expression/synthesis of PTH.

Question 46

Actions of vitamin D in calcium homeostasis

Answer 46

• Increases Ca2+ absorption in the gut (its primary action).
• It synergises with PTH on bone, causing osteoclasts to become active (via osteoblast releasing RANK ligand).
• Inhibits PTH synthesis, as a regulatory process (if you are absorbing more calcium from gut not as much is needed from the bone).
• Inhibits 1-alpha hydroxylase (self regulating -ve feedback to prevent hypercalcemia from too much vitamin D).

Question 47

Describe transport of calcium across the epithelial cells of the intestine. What is the role of vitamin D in this process?

Answer 47

• There is the paracellular route where calcium leaks through the tight junctions between cells and this isn’t regulated really.
• At the apical side, calcium enter into the gut cell via a selective calcium transporter (TRPV) and then binds to calbindin which transports calcium across the cell and allows it to be extruded on the basolateral membrane by Ca2+ ATPase or 3Na+/Ca2+. This is the transcellular route.
• Vitamin D acts on the vitamin D receptor which modulates (increases) the gene expression of TRPV and calbindin which increases the proteins that actively transport calbindin from the intestine into the body.

Question 48

Why can some tissues that are not the skin synthesis vitamin D and give some examples?

Answer 48

Several tissues are also able to locally synthesise vitamin D (1,25(OH)2D) from circulating 25(OH)D because they have the 1-alpha hydroxylase enzyme.
Such tissues/cells include macrophages and monocytes, keratinocytes, breast tissue, parathyroid, colon and placenta.

Question 49

Where are vitamin D receptors found?

Answer 49

Vitamin D is important for differentiation and its receptors are found in more than 30 different cell types e.g. keratinocytes in the skin, lymphocytes, macrophages, adipocytes, pancreatic beta cells, cells of breast, testis, ovaries, prostate, colon etc.

Question 50

Role of vitamin D in different tissues

Answer 50

Intestine: Increase in calcium and phosphate absorption.
Bone: Increased mineralisation mobilises calcium scores.
T and B lymphocytes: Immunomodulation (prevention of autoimmune diseases).
Muscle: Increases muscle strength.
Cancer: Regulation of cell growth so cancer prevention.
Muscle: Increased muscle strength.
Pancreatic cells: Decreased insulin resistance,
Heart: Prevention of CVD by modulating RAAS.

Question 51

Describe role of Klotho in the FGF23 receptor

Answer 51

Klotho is a single transmembrane protein which serves as an obligate co-receptor enabling FGF23 to interact with its receptor and it is also the modifier dictating which tissues respond to FGF23.

Question 52

Where is Klotho mostly expressed?

Answer 52

• Predominantly in distal convoluted tubules
• Epithelium of the choroid plexus in the brain
• Parathyroid glands

Question 53

Describe endocrine response to vitamin D deficiency

Answer 53

• Vitamin D deficiency leads to decreased Ca2+ intestinal absorption so decreased Ca2+ in the blood.
• The response to this is to increase PTH.
• This causes increased Ca2+ reabsorption from the kidney and also increased bone resorption to try raise the serum Ca2+ back to normal.

Question 54

When is FGF23 produced?

Answer 54

FGF23 is produced in the bone in response to high phosphate. FGF23 increases excretion of phosphate from the kidney and inhibits the activation of vitamin D (by inhibiting 1-alpha hydroxylase) to stop absorbing phosphate from the gut. So FGF23 reduces serum phosphate concentrations.