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) Calcium ions Extracellular Concentration total, in serum 2.5 x 10 3 M free 1.2x10"3M Functions Bone mineral Blood coagulation Membrane excitability Phosphate ions 1.00 x 10 3 M 0.85 x 10"3M Bone mineral Intracellular Concentration 10 7 M 1-2 x 10 3M Functions Signal for: • Neuron activation • Hormone secretion • Muscle contraction "A • Structural role • High energy bonds • Regulation of proteins by phosphorylation Extra- and intracellular calcium Extracellular calcium - Cartilage and bone mineralization - Cofactor of enzymes including proteins of coagulation cascade „Source" of intracellular calcium Excitable tissues Intracellular calcium - Signaling role - Contractility - Excitability - Neurosecretion - Endocrine and exocrine secretion - Cell differentiation and proliferation - Cell death and its regulation Calcium and its intake Calcium absorption - 25-60 % - Age - Dietary habits and calcium content in diet - Bone tissue requirements - Vitamin D Stomach - Gastric juice and role of HCI - Signalization connected to HCI production Small intestine - Duodenum a jejunum-90 % - Adaptive intake - duodenum and ileum < 50% daily intake absorbed 2 fractions or poofs • Readily exchangeable * Bone ECF for [Ca2+] regulation • Stable: bone remodeling 850 rng/d Constant intestinal loss Bane Kidney ^99% filtered ca reabsorbed 40% PTH-regulated Minimal urinary loss Bone tissue is crucial calcium and phosphorus storage tissue. Age-related negative calcium balance is an osteoporosis risk factor. Mechanisms of calcium absorptio 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? it. Estradiol Lumen Microvilli Actin Myosin-I Calmodulin O Ca2t O Ca2* O Exocytosis Lamina propria Calcium on blood (calcemia) o u ionised calcium (50%) A Acidosis Alkalosis Protein bound {40%) * 80-90% albumin: pH dependent * Acidosis I binding & T ionized Ca2+ - Alkalosis T binding & i ionized Ca2+ * 10-20% globulins Completed <10%) * Citrate & phosphate Compfexed & ionized Ca++ cross plasma membrane 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 Calbindin-D28K Luminal membrane Ca2+/Na+ exchanger Interstitial space Basolateral membrane Phosphorus Distribution - Bones cca 45 % - Ca10(PO4)6(OH)2 - Organic and inorganic form in ICF and ECF Age, sex, growth Blood - Concentration 1 mM (serum) - Ionized form (HP042-, H2P04) - 12 % protein complexes - Intracellular concentration approximately same as extracellular - Cotransport with sodium :unctions Structural - NA, phospholipids Modified saccharides, phosphoproteins, cofactors, G proteins Macroergic compounds (ATP) Regulatory role - signaling cascade, energetic processes Kidneys Reabsorption - proximal tubule (85 %) - Nptl-3 1,25 o enterocyt O-fTRPMt|—► ggj O-tTRFMÖ^-^O'' ? (^Na* O *4-H 1- o o o K*0 < -O NaVK' ATPase ■ Collagen type I = most important protein of bone matrix Bone matrix and bone minera Type I collagen monomeric and fibrillar structure B Overlap zone Gap zone N U I LU U LLU U I LU U Lili edc b aedc ba - Trpte mim kMM X 1 - C-mopUtme -oteulLir Sccrelon Kim lUlt* ^1 —i I (— Mm* í fornutnn Trombospondin Fibronectin Matrix Gla protein Osteocalcin Biglykan Decorin Bone sialoprotein Osteopontin Osteoadherin CO era' 5' 00 3 QJ i-+ O T3 O n>" l/l 0 Vitamin K-dependenty carboxylation and phosphorylation •a Ca affinity and mineralization Collagen and its synthesis I Secretion Procollagen Procollagen—I N-proteinase N-propeptides P1NP Propeptide cleavage I— Procollagen C-proteinase t N-telopeptides Procollagen C-propeptides P1CP Fibril formation C-telopeptides Catheps --------- in K NTX-1 B CTX-1 Pyridinoline (Pyr) Deoxypyridinoline (DPD) ICTP or CTX-MMP MMP Collagen synthesis Collagen degradation Collagenases- MMP - MMP1 and MMP13 - osteoblasts - Role of hormones and cytokines Mineralization production of small hydroxyapatite crystals (Ca, phosphates, carbonates, Mg, Na, K) Endopeptidases, PHEX- FGF23 External mechanism - alkalic phosphatase Internal mechanism - phosphol (Phosphoethanolamine/ phosphocholine phosphatase) Deposition of calcium Ca, P, and AF availability Vesicle formation (matrix) Collagen and its arrangement Cleavage of pyrophosphate Phosphate availability for mineralization Diet, calcium in diet, calcium/phosphorus in ECF Bone tissue and its remodeling Modeling versus remodeling of bone tissue REMODELING UNIT - BMU osteoblasts I osteoclasts osteocytes OCL Bone lining cells Lining cells Bone reabsorption Building of bone tissue osteoclasts mononuclear cells preosteoblasts asts 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 non-collagen 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-ß Mineralized bone Osteoclast Osteoblasts Osteoid Empty lacuna Bone-lining cells Osteocytes Lining cells - Stimulation of OB differentiation - OC communication - Differentiation to OB stimulated by PTH Osteoclasts (OK) - Bone tissue reabsorption Osteoclasts Prostaglandins, multiple hormones, cytokines, ILs and vitamin D Glucocorticoids - indirect inhibition of bone resorption Key factor regulating bone resorption is RANKL/OPG ratio. Production of mixture of pro- and anticlastogenic factors (differences in time) 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 Expression of different I receptors in time (effect of various stimuli) Osteoclastogenesis (+) RANKL (-) OPG HSC Stromal/ osteoblastic cells cytokines U OPG, RANKL, M-CSF TNF, IL-1, IL-6, IL-7, other ILs J IFN RANKL TNF 0®-®- -A M-CSF M-CSF W^n^s-^/ c-Fms-RANK- C-Fms+ RANK- c-Fms+ RANK+ M-CSF + RANKL T OPG Bone tissue resorption by osteoclasts ^ avp3 Integrin Lysosomes Cat K H CIC-7 f V-ATPase Ý RANK/RANKL Role of compartmentalization in bone resorption - podosomes Calcitonin and receptor f^eľc Resorption and secretion of bone resorption products - transcytosis Essential role of pH for bone tissue resorption Factors affecting bone tissue remodeling Resorption takes approx. 2 weeks Mineralization and formation approx. 12 weeks In pathophysiologic conditions is disrupted the continuity of bone tissue resorption and formation. Prostaglandins Cytokines - IL-la, IL-lß, Cytokines - IL-4, IL-13, IL TNF-a, TNF-ß, 10, IL-18 proinflammatory IL {7, IS, 17) TGF-a and EGF, FGF21, VE^^^^^1 ^^23 IGF-1 (endo-/paracrine) Prostaglandins h h BMPs (OB, autocrine) Immediate calcium need - homeostasis Ensuring mechanical requirements Endocrine regulation of bone tissue PTH Vitamin D Calcitonin Growth hormone IGF-1 Glucocorticoids Thyroid hormones Insulin Sex hormones Stimulation of resorption (long-term effect) Stimulation of bone formation (pulsatile effect) Stimulation of local secretion of IL-1 and IL-6 Stimulation of resorption (higher concentration) Inhibition of mineralization (higher concentration) Stimulation of bone formation (low concentrations, with PTH) Inhibition of resorption Regulation of bone tissue remodeling Stimulation of bone turnover Stimulation of osteoblast proliferation and differentiation Increased synthesis of collagen and other proteins 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 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 Stimulation of bone tissue formation and mineralization Increased collagen synthesis Stimulation of IGF-1 secretion Epiphyseal closure (E) Inhibition of RANKL secretion Changes in speed of bone resorption and formation (stimulation of formation and mineralization) Osteoblasts, lining cells, osteocytes Osteoblasts (primarily) Osteoclasts Osteoblasts - primarily GH Osteoblasts and osteoclasts IGF-1 Osteoblasts, osteocytes, osteoclasts Osteoblasts, osteoclasts (also indirect through TSH) Primarily osteoblasts Primarily osteoblasts, also other bone cells Prolactin Indirect effect insulin nsulin - osteocalcin axis Insulin o o o O O o o 0o ° Pancreas f Insulin sensitivity Adipose tissue Osteoblast —o o o o o leptin Activated osteoclast Osteocalcin RANKL :hydroxyapatite Undercarboxylated osteocalcin Mineralized bone Acidic resorption pit Decarboxylated OCN (Glul3-0CN) Bone tissue metabolism markers Ionized calcium Plasmatic phosphates PTH Vitamin D 8.5-10.5 mg/dL 3-4.5 mg/dL 10-65 pg/mL 30-100 ng/mL Marker Tissue origin Analytical sample Analytical method Hydroxyproline, total and dialyzable (OH-Pro, OHP); specific for all fibrilar collagens and a part of collagen proteins, including Ciq and elastin; present in newly synthesized and mature collagen bone, skin, cartilage, soft tissues urine colorimetry, HPLC Pyridinoline(PYD, Pyr); high concentrations in cartilage and bone collagen: not present in skin; present only in mature collagen bone.tendon, cartilage urine HPLC, ELISA Deoxypyrindoline (DPD, d-Pyr); high concentrations only in bone collagen: not present in cartilage or in skin; present only in mature collagen bone, dentine urine HPLC, ELISA Cross-linked C-terminal telopeptideof type 1 collagen (ICTP); high proportion from bone collagen in type 1 collagen; can partly originate from newly synthesized collagen bone, skin serum RIA Cross-linked C-terminal telopeptide of type 1 collagen (fragments alpha-CTX, beta-CTX); in type 1 collagen; probably high proportion from bone collagen all tissue containing type 1 collagen urine, serum ELISA, RIA, ECL1A Cross-linked N-terminal telopeptide of type 1 collagen (fragments NTX); in type! collagen; big proportion from bone all tissue containing type 1 collagen urine (alpha/ beta), serum (only beta) ELISA, RIA, IC MA Hyd roxy 1 ysine-gly cosid es (Hyl -Glyc); collagens and collagen proteins; glucogalactosyl- hydroxilysine is highly represented in soft tissue collagens and Clq; galactosil-OHLys is highly represented in bone collagen bone, skin, soft tissue, serum complement urine HPLC, ELISA Bonesialoprotein (BSP); synthesized by active osteoblasts and lay in extracellular bone matrix; it seems to express osteoclast activity bone, dentine, hypertrophic catrilage serum RIA, ELISA Tartarat-resistant acid phosphatase (TR-ACP); osteoclasts, thrombocytes, erythrocytes bone, blood plasma/serum colorimetry, RIA, ELISA Free gamma carboxyglutamin acid (GLA); resulted from bone proteins (e.g. osteocalcin, matrix Gla protein) and from coagulation factor blood, bone serum/urine HPLC HPLC - high performance liquid chromatography; ELISA - enzyme-linked immunosorbent assay; RIA - radio immuno assay; ECLIA - electrochemiluminiscence immunoassay; ICMA - immunochemiluminometiic assay Clinical relevance • Osteogenesis imperfecta • Osteopetrosis • Osteomalacia • Rachitis • Osteopenia - T score -1 - - • Osteoporosis - T score und 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 Minimal secretion Cell proliferation of chief cells is an important adaptive mechanism for: Hypocalcemia Low levels of vitamin D(l,25(OH)2D3) Hyperphosphatemia (uremia) Neoplastic growth 1,10,,7S 21 28 V6 14 16 I 9 10 11 12 13 14 15 16 17 Plasma calcium mg/dL T 4 Plasma PTH (arbitrary units) .evel of ionized calcium in blood is a key parameter for PTH secretion. 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. Calcium sensing receptors - CaSR - and PTH secretion o CU caidum + amino acids, peptides, Mg r Inhibition of cell growth PIP,-- DAG Kinass phosphorylation ] _> CaJcium (3 qC-—J ' ■-- 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 O PTH Main effects of PTH proximal Tubule H,PCa, P04 excretion Effects: (+) stimulatory (-) inhibitory ■ i bidirectional) ► fluxes--- binding effects [#]dlfferentlal equation numbeA Ca = calcium. ECF Ca = extracellular fluid Ca. OC = osteoclast. OC|m=OC precursor. OB = osteoblast. 0P6 = Osteoprotegerin. PO, = phosphate. PTH ■ parathyroid hormone. RANK = receptor of NF-Kappa 8. RANKL = RANK \Llgand, ROB = responding OB, TGFß = transfonrtlng growth factor beta, 1 -o-OH = t alpha hydroxylase