Ca, Phos, PTH

Question 1

Factors stimulating FGF release
Where does FGF23 come from

Answer 1

Made by osteocytes

Production is increased by stimulation from Ca/Pi bingin calciprotein receptors or by PTH or calcitriol effects.
It is inhibited by Mg

There is a delay from stimulus to effect as needs to be made by the osteocyte

Question 2

Organs involved in phosphate homeostasis and calcium homeostasis

Answer 2

Phosphate homeostasis involves four organs, which interact via the action of the hormones PTH, FGF23, and calcitriol. Osteocytes and osteoblasts secrete FGF23, while proximal tubular cells convert inactive 25(OH)2 vitamin D to active calcitriol via the action of the enzyme 1 alpha-hydroxylase [3]. These endocrine hormones regulate the expression and function of tissue-specific phosphate transporters

Question 3

Factors affecting PO4 absorption from GIT

Answer 3

• Digestibility: inorganic Pi can be much more available for absorption compared to natural
• Ca:P ratio inversely affects P availability for absorption
• High P in diet increases paracellular diffusion
• Vit D increases active absorption via Na/PO4 transporters.

Question 4

Factors regulating renal excretion of PO4

Answer 4

In healthy subjects, nearly 100% of sPi is filtered via the renal glomerulus and 80% to 90% is typically reabsorbed via sodium-mediated facilitated cotransporters in the renal tubule
- amount of Pi entering tubule is dependent on GFR, it cannot be added by the tubule (so if reduced the excretion rate decreases)
- PTH increases expression of Na/PO4 transporter increasing phos excretion
- Vit D increases reabsorption
- Acidosis increases excretion of PO4 to remove H+ (and alkalosis does the opposite)
- FGF23 increases PO4 excretion but is reliant on renal derived alpha-klotho which enables FGF23 to bind its receptor

Question 5

Effect of Dietary PO4 on homeostasis - JVIM 2020 impact of dietary PO4 in cats

Answer 5

Benefits: urine alkalinisation, prevent dental disease and aid in processing of food
No current upper limit guidelines, but known that inorganic PO4 can affect renal health parameters.
–> excess inorganic PO4 can damage kidneys particularly if inverse Ca:PO4 ratio

However, Lack of control of the Ca : P ratio is a common confounding issue in studies investigating the effects of high P intake
AND: There is currently no evidence that P in commercial cat foods induces renal disease
- though a study found association between high PO4 in diet and presence of CKD in cats (not dogs) based on owner questionnaire (where home made diets were the predominant fed diet).

Multiple studies show adverse renal effects in cats fed diets containing highly soluble inorganic Pi especially, but not exclusively, in diets with a Ca : P ratio less than 1 : 1

Question 6

Major organs and hormones of Ca homeostasis

Answer 6

parathyroid glands, kidneys, small intestine and skeletal bone

PTH, PTHr, Vit D (calcitriol)

Calcitonin (opposes)

Question 7

Functions of PTH

Answer 7

Increase blood Ca concentration

Increase tubular reabsorption of Ca
- Direct action on the distal convoluted tubule
- Indirectly in the ascending thick loop of henle, by increasing lumen net positive charge creating a stimulus for diffusion out of the nephron.

Increased bone resorption and number of osteoclasts
- Receptors on osteoblasts stimulate Ca release from bone and direct an increase in osteoclastic bone resorption
- Response is biphasic with the rapid effect being dependent on continuous presence of the hormone. Occurs through action of an osteocyte-osteoblast pump

Accelerate the formation of active vitamin D (1,25-dihydroxyvitamin D), inducing synthesis and activity of the mitochondrial enzyme in renal tubular epithelial cells. Increase in Vit D enhances SI absorption of calcium

Question 8

Production of Vit D and its regulators

Answer 8

reliant on their diet to obtain vitamin D
Vitamin D is available in two forms, namely vitamin D2 (ergocalciferol) and D3 (cholecalciferol)

enter the circulation and are predominately bound to the vitamin D binding protein (VDBP), with a small percentage also bound to albumin
–> some tissues express VDBP R that allows uptake and use before conversion

Vitamin D2/3 are prohormones that are subsequently activated by sequential hydroxylation steps by the action of cytochrome P450 (CYP) enzyme family in the liver
regulation of CYP27B1 is tightly controlled via parathyroid hormone (PTH) and FGF23 as well as negative feedback from calcitriol which inhibits the enzyme

Hydroxylation at C1α in the proximal tubule of the kidney (in mitochondria) converts 25(OH)D2/3 to the most hormonally active form –> calcitriol

PTH, calcitonin and hypoCa directly stimulate calcitriol production

Question 9

Effect of FGF23 on Vit D

Answer 9

Inhibits conversion of 25hydroxy D3 to calcitriol in the renal tubule.
–> further impairing renal reabsorption of PO4

HyperCa; FGF-23 and Pi loading all inhibit D3 production

Question 10

Actions of Vit D

Answer 10

Binds to VDR (wide expression) –> heterodimerises with the retinoic acid receptor, retinoid X receptor (RXR). This complex exerts genomic actions as a transcription factor to regulate target genes that contain a vitamin D response element in their promoter

Can also bind plasma membrane VDR to exert rapid responses: such as increased intestinal absorption of Ca and increased renal tubule Ca reabsorption and increase release of Ca from bone (Necessary for bone resorption because it promotes differentiation of monocytic haematopoietic precursors in the bone marrow into osteoclasts).

Also maintains GI barrier function - upregulates tight junctions and stimulates production of brush border enzymes and through suppressing tumour necrosis factor-alpha and nuclear factor kappa-beta pathways

Supresses tissue fibrosis through inhibition of TGF-B
Antiproliferative effects

Question 11

Factors affecting phosphorus absorption from GTI

Answer 11

P absorption in most species is dependent on the intestinal pH, P needs of the animal, source of P, and interactions with other dietary factors such as dietary Ca, magnesium (Mg), and phytates

Dietary Ca and Ca:P ratio exert an influence on P availability, with intestinal absorption of P inversely affected by dietary Ca and Ca : P ratio

Question 12

Source and stimulus of calcitonin

Answer 12

Synthesised by thyroid C cells in response to increased Ca

Major site of action is in the bone where it inhibits osteoclastic bone resorption. These are transitory effects and have a relatively minor role in homeostasis. With excessive production there is no disruption of homeostasis

Enhances calciuresis

Question 13

Stimulus for PTH release

Answer 13

Decreased Circulating Ca levels (CasR) - doesnt completely stop even in states of hypercalcaemia (though Ca will escape PTH action sin kidney and be excreted)

Increased PO4 levels

Vit D slows production of PTH

Question 14

Response to hypocalcaemia (acute and chronic)

Answer 14

Marked increase in PTH secretion (and gene transcription) and reduced breakdown of PTH in circulation extending its half-life.
Renal calcium reabsorption is increased, phosphorus excretion is increased within minutes
Bone Ca/P mobilisation occurs within 1-2 hours.

After several hours of high PTH/hypocalcaemia the production of calcitriol in the kidneys is increased
Increased absorption of intestinal Ca/P

With chronicity the production of PTH by the parathyroid gland is increased through hypertrophy/hyperplasia.

Question 15

Response to hypercalcaemia

Answer 15

reduces secretion of PTH from parathyroid chief cells and enhances intracellular degradation of the protein.
Increased calcitonin secretion is stimulated to minimise magnitude of hypercalcaemia, results in hyperplasia of thyroid C cells.

This mechanism is insufficient to control hypercalcaemia due to the transient nature of effects
Calcitriol synthesis is decreased through reduced PTH stimulation and direct inhibition of iCa on production.

Question 16

Types of Rickets and consequences

Answer 16

Provisional diagnosis of these congenital vitamin D disorders is usually based on compatible clinical signs in young patients, who have a dietary history that demonstrates adequate vitamin D intake. Definitive diagnosis is more challenging

Type 1A Vit D dependent rickets is characterised by deficiency in the 1alpha-hydroxylase CYPP27B1 enzyme in renal tissues thus Vit D is not converted to active calcitriol

Type 1B - mutations in CYP2R1 gene leading to failure of vitamin D to be converted to 25(OH)D in liver

main consequences of these disorders are hypocalcaemia which can be severe enough to cause seizures and skeletal abnormalities, generalised skeletal pain can also be a feature

Type 2A is characterised by end organ resistance to calcitriol and isolated incidences have been reported (hypoCa with elevated PTH)

Question 17

Systemic effects of hypercalcaemia

Answer 17

Systemic Effects:
- Renal: antagonise ADH, inhibits NaCl reabsorption → NDI
→ Polyuria, polydipsia: down-regulates ADH release and inactivates downstream signalling.
→ mineralisation of tubules and basement membranes and interstitial fibrosis, enhanced by RSHPTH (as increases Ca entry into tubular cells where it exerts toxic effects)
→ reduced ECV from GI signs → ischaemia and AKI
- GI - Vomiting, anorexia - reduced excitability of GI enteric NS and hyperacidity +/- ulceration
→ exacerbation of hypovolaemia and pre-renal azotaemia
→ may also see constipation (reduced motility, dehydration)
- Urolithiasis: reported in up to 31%, excessive calciuresis and PO4 excretion.
- Hypercalcaemic crisis- multi organ failure
- Soft tissue mineralisation (if Ca x PO4 > 60)
- Muscle twitches from membrane hyperexcitability and Arrhythmias: ECG: prolonged P-R interval most common from prolonged impulse

Question 18

Clinical findings in hyperPTH

Answer 18

Increased iCa
JSAP 2020 - >33% of dogs with normal tCa will have elevated iCa based on study of 65 dogs. And not always associated with low albumin
Though high tCa was strongly predictive of elevated iCa in another study.
Increased PTH (or inappropriately high normal 73%)
Chemiluminescent assay not recommended, instead use immunoradiometric (JSAP 2019)
DECREASED/Normal PO4
Normal PTHrP
Renal azotaemia is rarely present, and because renal function is normal PO4 is not increased
ECG: prolonged P-R interval
U/s - can assess for evidence of nodule (usually <4mm but there is wide variation). IF secondary disease then tend to see multiple nodules

Question 19

DDx for HyperCa

Answer 19

• Haematopoietic malignancy: look in BM/blood smear, nodes
• PTHrp paraneoplastic: measure blood levels (where available), usually PTH is low. Imaging to look for masses/bony lysis, anal sac palpation
• Hypoadrenocorticism (USUALLY MILD)
• Granulomatous disease → macrophage calcitriol synthesis (infectious disease testing for fungal, protozoal)
• Toxicity (grape)
• Osteomyelitis
  With secondary hyperparathyroidism (renal or nutritional) Ca is often normal/low with variable Pi and increased PTH but u/s of gland finds multiple nodules

Question 20

Medical Tx of hyperCa

Answer 20

Supportive: IVFT enhances calciuresis, frusemide, calcitonin
Glucocorticoids: work best for haematopoietic malignancy associated.
Increase excretion, reduce intestinal absorption and bone resorption

Bisphosphonates - bind to hydroxyapatite and inhibit osteoclast activity and induce osteoclast apoptosis
- Zoledronate: more potent antiresorptive effects than pamidronate. Causes increased urinary Ca and Pi excretion.
AEs: azotaemia, allergic reactions, hypoCa, vomiting, osteonecrosis
→ case series of 95 dogs (10 with hyperCa, remainder bone tumours) reported similar AEs. Initial efficacy in hyperPTH cases.
- Alendronate: AEs: poorly reported but include GI upset, hypoCalcaemia

Calcitonin: reduces osteoclast synthesis and activity → reduced bone resorption. Short duration of effect and resistance develops. Used in acute setting

Cholestyramine - sequesters Vit D in bile. used in toxicity

Question 21

Possible causes of idiopathic hypercalcaemia in cats and clinical findings

Answer 21

acidifying diets, urine acidifiers, chronic metabolic acidosis → increased bone resorption
Reduced response to calcitonin (reported in small experimental study)
Renal Diets - can cause and treat hypercalcaemia in cats

Increased iCa (mild to moderate) with normal/low PTH ( if can be measured), normal (or mildly increased) Pi and normal PTHrp and Vit D. JFMS 2016 review

NB low PTH can be a normal finding in cats due to insensitivity of assay

Question 22

DDx for hypoCa

Answer 22

Hypoparathyroidism - rare, presumed immune mediated destruction

DDx: hypoalbuminemia, insufficient dietary intake, eclampsia.

CKD - reduced Vit D synthesis and reduced active FGF23
→ reduced GI absorption and increased renal excretion
→ RSHPTH (tends to affect bones of face → rubber jaw)
Critical Illness - may be caused by proinflammatory cytokines,

Dietary Ca or Vit D deficiency

Acute Pancreatitis - sequestration to peripancreatic fat

Eclampsia (1-3 weeks post partum)
SI disease - reduced Vit D absorption, reduced Ca absorption (often have low Vit D and high PTH)
→ Nutritional secondary hyperPTH → vertebral osteopaenia, myelopathy

Hungry Bone Syndrome- occurs after correction of hyperPTH → rapid uptake by bone due to depleted stores
HAC - GC suppress intestinal absorption of Ca and increase renal excretion. Often PTH is increased.

Acute hyperMg or HypoMg- functional reduction in PTH as release is suppressed and increased resistance to its functions

Question 23

Clinical findings in hypoCa

Answer 23

Neurological signs are most common presenting complaint and worsen with exercise induced alkalosis.
Facial pruritus; muscle fasiculations, cramping, stiff gait
Behavioural: agitation, aggression
Seizures, tachycardia, hyperthermia, tense abdomen

ECG: prolonged Q-T interval due to effects on Ca mediated L type Ca channels

Question 24

Deactivation of Vit D

Answer 24

Enhanced by FGF23 from osteocytes the breakdown products can be active and can undergo C3 epimerisation which also generates active metabolites.

Metabolites are also bound to VDBP in circulation

Question 25

Primary Vit D metabolites measured in Vet med - JSAP 2021 review

Answer 25

25(OH)D (calcidiol) and 1,25(OH)2D (calcitriol) 25(OH)D has a half-life of 2 to 3 weeks and is widely regarded as the most accurate measurement to assess vitamin D status. It serves as a reservoir for the generation of the more biologically active 1,25(OH)2D (calcitriol)

Question 26

Difficulties with Vit D measurement

Answer 26

wide range of assays including chemiluminescent immunoassay, enzyme immunoassay, radioimmunoassay, high-performance liquid chromatography (HPLC) and the widely considered gold standard technique of liquid chromatography tandem mass spectrometry (LC–MS/MS) One of the main challenges regarding vitamin D analysis in both human and veterinary samples is the lack of standardisation between laboratories Immunoassay-based techniques: high throughput ; good sensitivity for 25(OH)D and require minimal sample volume BUT selectivity continues to be one of their major limitations. Cross-reactivity with different vitamin D metabolites, especially 24,25(OH)2D,unequal cross-reactivity of the two metabolites can cause bias and have a significant impact depending on the sample being analysed LC–MS/MS is considered the gold-standard method, it has the capability to detect and quantify multiple, highly similar analytes simultaneously within one sample, enabling the profiling of several metabolites of the vitamin D pathway. ELISA - measures free 25(OH)D Further studies are required to ascertain whether total or free 25(OH)D concentrations is the best assessment of vitamin D status in companion animals. This is likely to have important clinical implications.

Question 27

Acquired Vit D defiency clinical signs and causes

Answer 27

Deficiency - home cooked diet, reduced GI absorption (PLE, EPI, liver disease) hypovitaminosis D state may result in hypocalcaemia, secondary hyperparathyroidism and possible skeletal abnormalities, typically rickets in young animals and osteomalacia in older dogs and cats

Question 28

Classic skeletal changes of Rickets

Answer 28

mineralisation of physeal and epiphyseal cartilage with lesions typically involving the fastest growing bones such as the radius, tibia, metacarpals and metatarsals. Clinically this manifests as a stiff, lame gait, deformed limbs, pain on palpation of bones and muscle weakness. Radiographs - widening of the physeal growth plate, metaphyseal flaring, poor skeletal mineralisation and potentially pathological fractures

Question 29

Exogenous causes of Vit D excess and clinical consequences, TX

Answer 29

Diet: significant over-fortification of foodstuff with vitamin D can lead to debilitating hypercalcaemia in both dogs and cats Toxins: rodenticides, psoriasis medications Where there is known exposure to potentially toxic doses of vitamin D, immediate treatment involves induction of emesis followed by administration of activated charcoal, in conjunction with supportive and symptomatic care, typically involving intravenous fluid therapy Clinical disease occurs in hypervitaminosis D states when vitamin D metabolite concentrations become sufficiently increased to cause hypercalcaemia, resulting in clinical signs such as polydipsia, polyuria, lethargy and inappetence

Question 30

Endogenous causes of Vit D excess

Answer 30

classically occurs in patients with granulomatous diseases where a dysregulated immune response results in the excessive production of 1,25(OH)2D from 25(OH)D, by macrophages lacking negative feedback regulation This syndrome has been reported in dogs with sterile granulomatous lymphadenitis, granulomatous inflammation following placement of a biological implant, Angiostrongylus vasorum infections and Mycobacterium avium subspecies hominissuis infection In cats, multiple infectious diseases have been associated with this syndrome: blastomycosis, Mycobacterium infection, feline infectious peritonitis, Toxoplasmosis, Nocardia infection, Cryptococcosis and rhinitis caused by Actinomyces.

Question 31

Role of hypovitaminosis D in non-skeletal disease (GI, Pan, Liver, Infections)

Answer 31

The relationship between low 25(OH)D concentrations and the development or outcomes of non-skeletal disorders cannot be considered to be invariably causative and may, in fact, be due to reverse causation recent study which longitudinally tracked both markers of inflammation and 25(OH)D concentrations in dogs undergoing elective surgery showed that the post-operative increase in acute phase proteins is mirrored by modest declines in total, but not free, serum 25(OH)D concentrations. Suggests and acute phase response GIT - PLE Growing consensus that low concentrations of 25(OH)D play an important role. Possible also blunted PTH response due to low Mg. Vit D due to malabsorption, reduced intake, inflammation and possibly hypoMg (needed for activation of Vit D) --> may also contribute to worsening of disease due to loss of immunomodulatory properties Pancreas/Hepatic Dz - Dogs with weight loss and EPI were found to have significantly lower serum 25(OH)D concentrations than dogs with EPI and stable weight. Cause may include: long term dietary fat malabsorption; loss of adipose tissue associated with weight loss; on-going reduced dietary intake - dogs with acute pancreatitis which survived had significantly higher serum 25(OH)D concentrations than dogs who died - rickets has occasionally been reported in dogs with liver diseases likely as a consequence of impaired intestinal absorption of vitamin D Infections: status has been found to be lower in patients with a wide variety of infectious diseases Cardiac: lower in CHF and chronic (B2) valvular disease than earlier stages -> may ave been confounded by patient ages

Question 32

Vit D and the immune system

Answer 32

growing evidence that vitamin D metabolites can influence the canine immune cell function and phenotype ex-vivo, typically switching innate immune cells from a pro-inflammatory to a more anti-inflammatory response Several studies have shown an inverse relationship between biomarkers of inflammation and vitamin D status in companion animals. However - the widely reported negative association between vitamin D status and inflammation is not an absolute consensus, with one study reporting an increase in both c-reactive protein and 25(OH)D concentrations in racing sled dogs Several studies have identified a relationship between low vitamin D status and adverse clinical outcomes in hospitalised cats and dogs

Question 33

Vit D role n oncology

Answer 33

Several studies have shown that vitamin D metabolites have anti-proliferative effects on canine cancer cell lines in-vitro Dogs with three different types of cancer (mast cell tumour, lymphoma and osteosarcoma) all had altered vitamin D metabolism compared to the healthy control population However, other studies have failed to find a difference in vitamin D homeostasis in dogs with and without cancer including the observation that 25(OH)D concentrations were not significantly different between dogs with an osteosarcoma and control dogs

Question 34

Vit D and Renal disease

Answer 34

In dogs with chronic kidney disease (CKD), lower levels of serum 25(OH)D have been identified. Low concentrations of 1,25(OH)2D have also been reported in both cats and dogs with CKD. This is thought to be due to a combination of lower availability of 25(OH)D, reduced CYP27B1 (1α-hydroxylase) activity due to renal damage and urinary loss of vitamin D --> considered important in the development of secondary hyperparathyroidism in CKD 2 studies looked at supplementation and showed improvement in metabolite levels HOWEVER -the benefits of long-term 25(OH)D supplementation on the progression of CKD, and the patient’s quality of life, remained undetermined .