Control of calcium metabolism. Calcium and phosphorus homeostasis Primary elements of blood tissue are calcium (Ca) and phosphorus (P). - up to 65 % of bone weight - almost all Ca and P supply, half of supply of Mg in human body - Essential role of these elements in physiological processes Bone tissue - 99 % of overall Ca, of it 99 % in mineral component - 1 % - quickly mobilizable and convertible (ICF - ECF) Extra- and intracellular calcium Intracellular calcium - Signaling role - Contractility - Excitability - Neurosecretion - Endocrine and exocrine secretion - Cell differentiation and proliferation - Cell death and its regulation Extracellular calcium - Cartilage and bone mineralization - Cofactor of enzymes including proteins of coagulation cascade - „Source“ of intracellular calcium - Excitable tissues ICF ECF MIT ER cca 1mM cca 100 nM cca 100 mMcca 25 mM Ca2+-ATPase Ca2+ IK Na+/Ca2+ exchanger VDAC/ Ca2+-uniporter mNCX Calcium and its intake Stomach - Gastric juice and role of HCl - Signalization connected to HCl production Calcium absorption - 25 – 60 % - Age - Dietary habits and calcium content in diet - Bone tissue requirements - Vitamin D Age-related negative calcium balance is an osteoporosis risk factor. Small intestine - Duodenum and jejunum – 90 % - Adaptive intake – duodenum and ileum Mechanisms of calcium absorption Paracellular - Luminal electrochemical gradient - Integrity of intercellular connections - Claudins and their role in paracellular transport Transcellular - TRPV6 and associated proteins - Recyclation of TRPV6 - Alternative mechanisms? VitaminD Glucocorticoids Estradiol Prolactin + +- Adaptation to dietary calcium levels Calcium on blood (calcemia) GlomerularfiltrationYES Endocrinecontrol GlomerularfiltrationNO Calcium excretion - 98 % of filtered Ca is reabsorbed - 70 % proximal tubule - 20 % thick ascending limb of HL - 5 % collecting duct - 2 % urine - CaSR (TALH) - Paracellin-1 - PTH Phosphorus Blood - Concentration 1 mM (serum) - Ionized form (HPO4 2-, H2PO4 -) - 12 % protein complexes - Intracellular concentration approximately same as extracellular - Cotransport with sodium Distribution - Bones cca 45 % - Ca10(PO4)6(OH)2 - Organic and inorganic form in ICF and ECF - Age, sex, growth Functions - Structural – NA, phospholipids - Modified saccharides, phosphoproteins, cofactors, G proteins - Macroergic compounds (ATP) - Regulatory role – signaling cascade, energetic processes Vitamin D PTH IGF-1 FGF23 Kidneys - Reabsorption - proximal tubule (85 %) – Npt1-3 Magnesium Blood - 0.7 – 1 mM - Approx. 30 % in protein complexes - 15 % in phosphate and low molecular weight anion complexes - 55 % free Distribution - 1 mol - bones approx. 54 %, muscles and soft tissues approx. 56 % - ECF – 0.5 mM Cell - 95 % in ATP and similar molecules - Concentration 0.5 mM - Ion channels? Functions - Cofactor (glycolytic, kinase and phosphatase systems) - Stabilizing function (DNA, RNA, ribosomes) - Activator of ATP transporters - Neuromuscular excitability Kidneys - 95 % of filtered amount is reabsorbed - 15 % PT, 70 % cortical TAHL, 10 % DT - Regulation – magnesemia, calcamia, ECF volume enterocyt Bone tissue physiology www.creab.org - Human Body Anatomy - Online anatomy atlas. Viktoria Ruppel. 14. 3 2015 Trabecular (spongy) bone – cca 20 % - High surface-to-volume ratio - High metabolic activity - Nutrients diffuse from ECF to trabecules Compact (cortical) bone – approx. 80 % - Low surface-to-volume ratio, osteocytes in resting state - Haversian canals with concentric layers of collagen – osteons (Haversian systems) - Collagen matrix impregnated with bone mineral crystals - 20 x 3-7 nm, mainly hydroxyapatite Bone matrix and bone mineral Collagen type I = most important protein of bone matrix Trombospondin Fibronectin Matrix Gla protein Osteocalcin Biglykan Decorin Bone sialoprotein Osteopontin Osteoadherin Vitamin K-dependent g carboxylation and phosphorylation Ca affinity and mineralization Signaling+haematopoiesis Collagen and its synthesis Collagen synthesis Collagen degradation Cathepsin K MMP Collagenases – MMP - MMP1 and MMP13 – osteoblasts - Role of hormones and cytokines Mineralization = production of small hydroxyapatite crystals (Ca, phosphates, carbonates, Mg, Na, K) External mechanism – alkalic phosphatase Internal mechanism – phospho1 (Phosphoethanolamine/ phosphocholine phosphatase ) Cleavage of pyrophosphate Phosphate availability for mineralization Vesicle formation (matrix) Collagen and its arrangement Ca, P, and AF availability Diet, calcium in diet, calcium/phosphorus in ECF Deposition of calcium SIEBLINGs - Osteopontin, DMP-1 (OC) - Bone sialoprotein, MEPE Endopeptidases, PHEX – FGF23 Bone tissue and its remodeling osteoclasts mononuclear cells preosteoblasts osteoblasts Bone reabsorption Building of bone tissue Modeling versus remodeling of bone tissue REMODELING UNIT - BMU osteoblasts osteoclasts osteocytes Bone lining cells Bone tissue and its remodeling Osteocytes (OC) - Metabolic activity - PTH receptors - Communication with bone surface - Mechanic sensing - RANKL production - Direct degradation of bone tissue (osteocytic osteolysis) - Adaptive remodeling Osteoblasts (OB) - Bone matrix production - Production of collagen and noncollagen peptides + their orientation - Regulation by hormones, local factors and cytokines - Differentiation and further fate – apotosis, osteocytes, lining cells - „recruitment“ of other cells – IGF- 1, IGF-2, TGF-b Lining cells - Stimulation of OB differentiation - OC communication - Differentiation to OB stimulated by PTH Osteoclasts (OK) - Bone tissue reabsorption Remodeling unit - BMU 1. Stimulatory and inhibitory signals of osteocytes (oncostatin M - OSM, sclerostin, PTHrP) 2. Stimulatory and inhibitory signals of osteoclasts to osteocytes (TGF-b, IGF-1, cardiotropin-1, Sema4D – semaforin 4D, sfingosin-1 phosphate) 3. Signalling between osteoblasts (ephrinB2, EphB4, Sema3a, PTHrP, OSM) 4. Stimulatory and inhibitory signals between osteoblasts and osteoclasts and their derivatives (RANKL, Sema3B, Wnt5a, osteoprotegerin - OPG) 5. Signalling between haematopoietic stem cells and osteoblasts (macrophage-produced OSM, IL produced by T-cells, RANKL) Osteocyte origin Osteocyte - Changes in metabolic activity - Formation of „projections“ – communication - Communication with other osteocytes (syncytium – OC + OB) Osteoclasts cytokines Expression of different receptors in time (effect of various stimuli) Production of mixture of pro- and anticlastogenic factors (differences in time) Glucocorticoids – indirect inhibition of bone resorption E2 (estrogens) – inhibition of T cell activation= inhibition of RANKL and TNF-a secretion Sex hormones – regulation of osteoblasts and osteoclasts differentiation, including length of their life Key factor regulating bone resorption is RANKL/OPG ratio. Osteoclastogenesis (+) RANKL (-) OPG Bone tissue resorption by osteoclasts Role of compartmentalization in bone resorption - podosomes Resorption and secretion of bone resorption products - transcytosis Essential role of pH for bone tissue resorption Factors influencing bone tissue remodeling Osteoblasts Lining cells Osteoclasts Remodeling of bone tissue Immediate calcium need - homeostasis Ensuring mechanical requirements Trabecularbone Systemic signals Local signalsResorption takes approx. 2 weeks Mineralization and formation approx. 12 weeks In pathophysiologic conditions is disrupted the continuity of bone tissue resorption and formation. Cytokines - IL-1α, IL-1β, TNF-α, TNF-β, proinflammatory IL (7, 15, 17) Cytokines - IL-4, IL-13, IL- 10, IL-18 TGF-α and EGF, FGF21, FGF23 Prostaglandins Prostaglandins PDGF VEGFA, HIF-1a (+/-) IGF-1 (endo-/paracrine) BMPs (OB, autocrine) Endocrine regulation of bone tissue Hormone Effect Target cells PTH - Stimulation of resorption (long-term effect) - Stimulation of bone formation (pulsatile effect) - Stimulation of local secretion of IL-1 and IL-6 Osteoblasts, lining cells, osteocytes Vitamin D - Stimulation of resorption (higher concentration) - Inhibition of mineralization (higher concentration) - Stimulation of bone formation (low concentrations, with PTH) Osteoblasts (primarily) Calcitonin - Inhibition of resorption - Regulation of bone tissue remodeling Osteoclasts Growth hormone IGF-1 - Stimulation of bone turnover - Stimulation of osteoblast proliferation and differentiation - Increased synthesis of collagen and other proteins Osteoblasts – primarily GH Osteoblasts and osteoclasts – IGF-1 Glucocorticoids - Decreased absorption of Ca in GIT - Induction of osteoclastogenesis - Increased bone resorption (+ RANKL) - Suppressed remodeling of bone tissue - Induction of apoptosis in osteoblasts and osteocytes - Inhibition of IGF-1 synthesis Osteoblasts, osteocytes, osteoclasts Thyroid hormones - Children – Stimulation of mineralization and epiphyseal maturation - Adults – increased resorption - Chondrocyte growth and proliferation (permissive effect on growth hormone) - Increased transcription of collagenase and gelatinase Osteoblasts, osteoclasts (also indirect through TSH) Insulin - Stimulation of bone tissue formation and mineralization - Increased collagen synthesis - Stimulation of IGF-1 secretion Primarily osteoblasts Sex hormones - Epiphyseal closure (E) - Inhibition of RANKL secretion - Changes in speed of bone resorption and formation (stimulation of formation and mineralization) Primarily osteoblasts, also other bone cells Prolactin - Indirect effect Insulin – osteocalcin axis insulin Decarboxylated OCN (Glu13-OCN) leptin Bone tissue metabolism markers Ionized calcium 8.5 – 10.5 mg/dL Plasmatic phosphates 3 – 4.5 mg/dL PTH 10 – 65 pg/mL Vitamin D 30 – 100 ng/mL Clinical relevance • Osteogenesis imperfecta • Osteopetrosis • Osteomalacia • Rachitis • Osteopenia – T score -1 – -2.5 • Osteoporosis – T score under -2.5 Parathormone Characteristics - Parathyroid glands – chief cells - Synthesis and storage of PTH - Very quick secretion of PTH - Ability to proliferate during long-term stimulation PTH - Synthetized as pre-pro-PTH - Several types of secretion granules (PTH; PTH+cathepsin B, H) - Very quick metabolization (70 % liver, 20 % kidneys) – 2 min - Presence of several types of fragments - PTHR1, PTHR2, PTHR3 – G prot. PTH secretion Level of ionized calcium in blood is a key parameter for PTH secretion. Minimal secretion Maximal secretion (reserve capacity) During sudden decrease of ionized calcium is PTH secretion increased. Vitamin D decreases PTH secretion (inhibits expression and production of PTH), NOT during chronic hypocalcemia Phosphates stimulate production and secretion of PTH with delay. Cell proliferation of chief cells is an important adaptive mechanism for: - Hypocalcemia - Low levels of vitamin D(1,25(OH)2D3) - Hyperphosphatemia (uremia) - Neoplastic growth Calcium sensing receptors - CaSR - and PTH secretion CaSR – G-protein coupled receptor - Activation of PLC - Inhibition of cAMP production Various distribution in tissues – all tissues participating in calcium homeostasis - Parathyroid glands - Kidneys - Skin - GIT epithelium, enterocytes - G cells of stomach - CNS Clinical aspects - Mutation – inactivation/activation - familial hypocalciuric hypercalcemia (in.) - Familial hypoparathyroidism with hypercalciuria (ac.) - Calcimimetics – inhibition of PTH secretion + amino acids, peptides, Mg Main effects of PTH (+) calcium resorption - cTAHL, DT - transcellular and paracellular transport - TRPV5 and TRPV6 – Ca2+ inhibition - Calbindin-D28K - NCX1 and PMCA (+) phosphate excretion - PT and DT - Inhibition of resorption - NaPi cotransporters – internalization, degradation (+) activity of 1a-hydroxylase - PT (-) resorption of Na, water and bicarbonate – PT (-) Na+/K+-APTase (basolateral membrane) (+) gluconeogenesis – PT (-) GFR - podocytes Effect of PTH on osteoclasts is indirect. Pulsatile secretion stimulates osteoblasts, chronic continual osteoclasts. PTH and bone tissue physiology + differentiation of osteoblast precursors + secretion of paracrine and autocrine factors (IGF-1) (-) apoptosis (+) osteocytes – release of Ca, osteocytic osteolysis Clinical application – osteoporosis therapy Parathyroid Hormone-Related Peptide - PTHrP Characteristics - First as a peptide produced by tumors – endocrine effect – kidneys + bones - Also paracrine – local increase of Ca concentration - Later discovered in many tissues Functions - Calciotropic hormone - Fetal development - proliferation and differentiation - Lactation – (+) resorption of bone tissue without possibility to affect by Ca supplementation - Skin – proliferation and differentiation - GIT, bladder, uterus – (+) smooth muscles relaxation - CNS - neuroprotection - Para-/auto-/intracrine effect Calcitonin Characteristics - C cells of thyroid gland - Family of peptides (amylin, CGRPs, adrenomedulin) - Different distribution in various tissues - Secretion is determined by level of ionized calcium (CaSR) - Stimulation of secretion: - Glucocorticoids - CGRP - Glucagon - Enteroglucagon - Gastrin - Pentagastrin - Pancreozymin - β-sympatomimetics - Inhibition of secretion - somatostatin Function unclear Functions - Bone tissue - Inhibition of osteoclast motility and differentiation - Inhibition of osteoclast secretion - ATPase inhibition - Kidneys - Increased excretion of Ca – inhibition of resorption (Ca2+ ion channels – LS, Na+/Ca2+ - BM) - Skeleton development? - Skeleton protection during pregnancy? Clinical relevance - Osteoporosis therapy - Paget disease therapy - Treatment of pain (bones metastases) - ! Increased risk of cancer Calcitonin gene, mRNA splicing and posttranslational modificationsCcells CNSandneuronsingeneral Russell FA, King R, Smillie SJ, Kodji X, Brain SD: CALCITONIN GENE-RELATED PEPTIDE: PHYSIOLOGY AND PATHOPHYSIOLOGY. Physiol Rev 2014, 94(4):1099-1142. Peripheral sensory nerves Vasodilatation Calcitonin gene-related peptide - CGRP Neuropeptide – sensoric and integrative motoric functions Russell FA, King R, Smillie SJ, Kodji X, Brain SD: CALCITONIN GENE-RELATED PEPTIDE: PHYSIOLOGY AND PATHOPHYSIOLOGY. Physiol Rev 2014, 94(4):1099-1142. Vasodilatation induced by various mechanisms - G prot. - eNOS/NO CGRP - functions Vitamin D….hormone?...vitamin? Characteristics - Intake with diet or synthesized (UV) - In blood bound to VDBP and albumin - Very small free fraction 1,25(OH)2D – cca 0,4 % PTH, prolactin, calcitonin, GH (+) T3/T4, metabolic acidosis (-) Ca, phosphates, 1,25(OH)2D, FGF23 (-) Ketoconazole VDBP Estrogens (+) 1a-hydroxylase - Expression in various tissues - Keratinocytes - Placenta - Macrophages Different 1a-hydroxylase expression = local tissue homeostasis Different rate of feedback control Cartilage,small intestine Physiological effects of vitamin D VDR - High affinity to 1,25(OH)2D - Level of circulating 1,25(OH)2D - Heterodimer with RXR – coactivators, corepressors Non-genomic effects - Rapid increase of intracellular Ca concentration - PLC activation - Opening of some Ca ion channels - Required VDR presence Vitamin D and Ca absorption/reabsorption - (+) CBP, AP, Ca2+/Mg2+-ATPase - (+) TRPV6 – absorption (GIT) - (+/-) TRPV5 – reabsorption (kidneys) - Calbindin-9K - 1,25(OH)2D-inducible ATP-dependent Ca2+ pump - Na+/Ca2+ exchanger Parathyroid glands - Gene expression regulation - Cell proliferation regulation - (-) PTH gene transcription Bones and bone tissue - (-) collagen synthesis - (+) osteocalcin synthesis - (+) osteoclasts differentiation – osteoclastogenesis - (+) RANKL - Main function – ensuring the stability of the bone microenvironment for mineralization by the standard intake and availability of Ca and phosphates Muscle tissue - (+) uptake AAs - (+) troponin C - Phospholipids metabolism Vitamin D and immune system Macrophages Dendritic cells T cells Clinical relevance - Analogue of vitamin D without ability to cause hypercalcemia - Antiproliferative effect – treatment of cancer? - Synergy with cyclosporin B – rejection of transplantates - Suppression of PTH synthesis – 22-oxacalcitriol (hyperparathyroidismus) - Psoriasis (clinical trials) FGF23 – fibroblast growth factor 23 Clinical relevance: - Autosomal dominant hypophosphatemic rickets (ADHR) - Tumor-induced osteomalacia (TIO) - Klotho mutation - Prediction of chronic kidney failure prognosis Characteristics - New hormone? - Overexpression = hypophosphatemia and decrease of 1a 25(OH)D hydroxylation Functions - maintaining normophosphatemia and regulation of vitamin D metabolism - Decreased expression of IIa, IIb, and IIc (NPT) – phosphate transport - Increased expression of 24-hydroxylase – inactive form - Klotho = co-receptor Regulation - Phosphorus availability in diet (-) - Serum phosphorus - 1,25(OH)2D - iron FGF23 Pars convoluta PT Inhibition Npt2aCyp271b 1, 25(OH)2D phosphate Feedback mechanism between DT and PCT endocrine, paracrine Calcium homeostasis – still just a simplified model