Fig. 4.1
Bioactive vitamin D activates the vitamin D receptor (VDR), which interacts with a neighboring retinoic acid receptor. This complex then binds to the vitamin D response element (VDRE) in the PTH gene promoter and downregulates PTH transcription. From: Vimaleswaran KS et al. Interaction between allelic variations in vitamin D receptor and retinoid X receptor genes on metabolic traits. BMC Genet 2014; 15:37
The relationship between calcitriol and PTH gene suppression has been used to prevent and treat secondary hyperparathyroidism in patients with renal failure [9]. Unfortunately, despite increases in dosing, up to 20–30 % of hemodialysis patients treated with a nonselective vitamin D receptor activators (VDRA) show no decrease in serum PTH levels [10]. The underlying mechanism causing resistance has been investigated in animal and human models [11, 12]. In rats with renal failure, the VDRs possess only half of the DNA binding capacity compared to their control counterparts. In addition, the incubation of normal VDRs in a uremic plasma ultrafiltrate results in more than a 50 % loss of VDRE binding sites . Uremic toxins may alter or destroy the DNA-binding sites resulting in an inadequate compensatory response following calcitriol administration [12].
In humans, particular polymorphisms in the VDR gene (12q12.14) have been identified in patients with chronic kidney disease that may affect their response to intravenous calcitriol [10]. In general, a higher incidence of the b allele of the VDR BsmI gene has been reported in hemodialysis patients with secondary hyperparathyroidism [13]. In predialysis patients with mild-to-severe chronic renal failure, patients with the BB genotype have a greater reduction in PTH levels following administration of a single bolus of calcitriol, despite having corrected for calcium and phosphorous levels. The patients with BB genotype also show slower disease progression compared to patients with the bb genotype. The authors from this study concluded that patients with the BB genotype could remain on hemodialysis longer before requiring a parathyroidectomy [14, 15]. However, it is important to note that when the parathyroid gland specimens were removed from these patients, tissue culture analysis of PTH secretion patterns were not associated with the various VDR alleles and response to calcitriol [15, 16]. Thus, at present, the current level of evidence does not support adapting treatment algorithms according to a patient’s VDR allele status [10]. A challenge for the future will be identifying other transcription regulators and mechanisms that may serve as biomarkers or possibly even therapeutic targets to aid in the management of these patients.
Posttranscriptional Regulation by Calcium and Phosphate
The amount of PTH synthesized for translation is highly dependent on events occurring after transcription (Fig. 4.2). For example, dietary-induced hypocalcemia results in a ten-fold increase in PTH mRNA levels via posttranscriptional processes alone [17]. The amount of mRNA available for translation is highly predicated on the survival of the newly synthesized strands. In the 3′ UTR , evolutionary conserved domains are present that correspond to elements in the mRNA that are highly prone to degradation by cytosolic ribonucleases [18]. Importantly, the instability of these mRNA elements is not absolute. In the setting of hypocalcemia, cytosolic trans (non-DNA mediated) activating factors may bind to the cis (DNA mediated)-acting instability elements and protect the mRNA from subsequent degradation [17]. Additionally, serum phosphate, independent of changes in the serum calcium, causes decreased binding of the cytosolic proteins [17, 19]. This results in de-adenylation, de-capping, and subsequent degradation of the mRNA [18] (Fig. 4.3).
Fig. 4.2
(a) Cellular processing of mRNA . Nascent mRNA comprised of exons (E1 through E4) and intervening sequences (IVS) is processed in the nucleus by 5′-methyl capping, splicing, cleavage, and polyadenylation. In the cytoplasm, AU-rich element-binding proteins (ARE-BPs, blue box and red oval) bind to AREs within the 3′-region of RNA and stabilize or destabilize mRNA. Stabilized mRNA undergoes translation in ribosomes, whereas destabilized mRNA undergoes deadenylation, decapping, and degradation in exosomes or P-bodies. (Adapted from reference 130 with permission from the American Society for Clinical Investigation.) (b) Processing of mRNA-encoding PTH. Murine mRNA-encoding PTH is bound by ARE-PPs, which either stabilize or destabilize the mRNA. The ratio of activities of stabilizing/destabilizing ARE-binding proteins bound to mRNA-encoding PTH determines the half-life of the mRNA. KSRP is a mRNA-destabilizing ARE-BP for mRNA-encoding PTH that is active in its dephosphorylated state. The peptidyl-prolyl isomerase Pin 1 is responsible for the dephosphorylation of KSRP. In CKD, Pin 1 activity is reduced, and as a result less dephosphorylated (active) KSRP is available. Consequently, a stabilizing ARE-BP, AUF1, is active and mRNA-encoding PTH is degraded to a lesser extent, resulting in higher intracellular mRNA levels, more PTH synthesis, and secondary hyperparathyroidism. Abbreviation: P, phosphate. (Adapted from reference 130 with permission from the American Society for Clinical Investigation.) From: Kumar R, Thompson JR. The regulation of parathyroid hormone secretion and synthesis. J Am Soc Nephrol 2011; 22: 216–224
Fig. 4.3
Low dietary intake of calcium and phosphate decrease PTH mRNA levels. Weanling rats were fed control (0.6 % calcium, 03 % phosphate), low calcium (0.02 % calcium, 0.6 % phosphate) or low phosphate (0.6 % calcium, 0.02 % phosphate) diets for 14 days. Total RNA from thyro-parathyroid tissue from each rat was extracted and PTH mRNA levels determined by Northern blots. Each lane represents PTH mRNA from a single rat. From: Kilav R, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 1995; 327–333 [20]
Translation and Protein Processing
PTH mRNA encodes a pre- (or signal) sequence of 25 amino acids and a basic pro-peptide of 6 amino acids [21]. PreProPTH (115 amino acids) is first synthesized on ribosomes that are bound to the membrane of the endoplasmic reticulum [6, 22]. As translation proceeds, the polypeptide, rich in hydrophobic residues, is transported into the endoplasmic reticulum where two amino (N)-terminal methionines (MET) are cleaved by methionyl amino peptidase ([23]). As the polypeptide chain is translocated across the ER, further proteolytic cleavage of the remaining signal sequence occurs at the glycyl-lysyl bond, resulting in 23 more amino acids being removed from the PTH precursor [6, 23]. The formation of ProPTH from preProPTH is estimated to occur in less than a minute [24].
ProPTH is exported in vesicles that bud from the transitional ER and carry their cargo through the ER-Golgi intermediate compartment and then to the Golgi network. Following entry into the trans-Golgi apparatus, the basic pro-peptide directs cleavage of the pro-sequence (6 amino acids) from the N-terminal to produce mature PTH (84 amino acids) ([25]). PTH is then packaged into either cytoplasmic (for storage) or secretory granules. The entire parathyroid biosynthetic process is estimated to occur in less than 1 h [26].
Regulated Secretion and Degradation of PTH and Its Derivatives
PTH secretion is regulated predominantly by calcium sensing receptors (CaSRs) located on the surface of chief cells [18] (Fig. 4.4). The CaSR is a seven-transmembrane G-protein-coupled receptor that is highly sensitive to changes in serum calcium [18]. For instance, a decrease of less than 1 mg/dL in serum calcium can cause PTH secretion to double [27] (Fig. 4.5). Sudden and sustained hypocalcemia results in elevations in PTH within 1 min, peaks at 4–10 mins, and then steadily declines to approximately 60 % of its maximum concentration, despite sustained hypocalcemia [18]. In contrast, the rate of PTH secretion is greatly suppressed when the serum calcium exceeds 9–10 mg/dL [27] (Fig. 4.5).
Fig. 4.4
Signaling pathway by which extracellular calcium (Ca2+) binds to the calcium sensing receptor (CaSR). Through the association of the CaSR with the i-type heterotrimeric G protein, Giα, adenylate cyclase (AC) activity is inhibited and cyclic AMP (cAMP) concentrations decrease. Association of the CaSR with the Gqα subunit of q-type heterotrimeric G protein results in the activation of PLC that increases inositol (1,4,5)P3 and diacylglycerol (DAG) with attendant downstream effects, such as an increase in intracellular calcium that is mobilized from intracellular stores, and the activation of PKC. MAPK and PLA2 are activated by Gqα-dependent pathways with increases in MEK and ERK and an increase in arachidonic acid formation. From: Kumar R, Thompson JR. The regulation of parathyroid hormone secretion and synthesis. J Am Soc Nephrol 2011; 22: 216–224
Fig. 4.5
Approximate effect of plasma calcium concentration on the plasma concentrations of parathyroid hormone and calcitonin. Note that chronic changes in calcium levels of only a few percentage points can cause as much as a 100 % change in parathyroid hormone concentration. From: Hall J. Parathyroid hormone, calcitonin, calcium and phosphate metabolism, vitamin D, bone and teeth, in Guyton and Hall textbook of medical physiology. Chapter 79. 11ed. Philadelphia: Saunders-Elsevier: 2011. Philadelphia 955–972
Following exocytosis from the chief cell, the liver and kidney metabolize PTH into amino (N)- and carboxy (C)-terminal fragments, which are ultimately cleared by glomerular filtration. Chief cells also partially degrade PTH (1–84) and secrete both N- and C-fragments directly into the circulation [6]. Traditionally, the N-terminal portion of PTH has been thought to constitute the biologically active region [6]. Substitution or deletion of even one amino acid in the first N-terminal 34 amino acids significantly reduces the polypeptide’s functional activity and potential to interact with the type 1 PTH receptor (PTH1-R) [6]. PTH fragments with conserved sequences in the first 34 amino acids [i.e., naturally occurring PTH (1–37), synthetic analogue PTH (1–34], PTHrP) are capable of mediating any number of activities that are classically associated with PTH [28].
The C-terminal of PTH (the last 50 amino acids) was previously thought to be biologically inert after translation [29]. Evidence now suggests that C-PTH fragments interact with non-classical PTH receptors and exert biological effects that are independent and opposite to those of PTH (1–84) [30]. For instance, C-PTH fragments may bind to C-PTH receptors on osteoclasts and exert a direct antiresoptive effect on bone [31, 32]. A particular subset of C-PTH fragments, representing approximately 10 % of all C-PTH fragments, contains a partially preserved N-structure [33]. Compared to other C-PTH fragments, the N-truncated fragments, represented by the prototype PTH (7–84), may become increasingly important clinically because they have about a ten-fold greater affinity for the C-PTH receptor and inducing its antiresorptive effects [33]. Moreover, synthetic hPTH (7–84) has been shown to antagonize the calcemic effect of hPTH (1–84) and hPTH (1–34) in parathyroidectomized animal models, suggesting that the fragments may at least contribute to the PTH resistance commonly observed in the setting of renal failure [34, 35].
The relative concentrations of PTH (1–84) and C-PTH fragments are predominantly a reflection of the serum calcium and renal status of the patient. In the setting of normocalcemia and renal sufficiency, PTH (1–84) has a half-life of 2–4 mins and accounts for approximately 20 % of total circulating PTH [36]. In patients with impaired renal clearance, the half-lives of PTH (1–84) and other PTH fragments with conserved sequences in the first 34 amino acids are mildly elevated and range from 4 to 6 mins [37]. In the setting of hypocalcemia and hypercalcemia, the relative concentration of PTH (1–84) compared with total circulating PTH ranges as high as 33 % or as low as 4 %, respectively [36, 26]).
In contrast to PTH (1–84), C-PTH fragments have an inherently longer half-life that ranges from 10 to 20 mins depending on the renal status of the patient [36]. C-PTH fragments account for approximately 80 % of circulating PTH in normal individuals and upwards of 95 % as glomerular filtration rate decreases [36, 26]). Similarly, the C-PTH fragments with partially preserved N-structure, such as PTH (7–84), act like other C-PTH fragments, and their concentration relative to PTH (1–84) increases in hypercalcemia and decreases in hypocalcemia [35]. In the setting of poor renal clearance, the N-truncated C-fragments accumulate and may account for up to 50 % (compared to only 15–20 % normally) of intact PTH (iPTH) immunoreactivity [34].
This at least partly explains why iPTH monitoring demonstrates a relatively slower decline following subtotal or total parathyroidectomy in the setting of chronic renal failure . Commercially available iPTH assays (first and second generations) for intraoperative iPTH monitoring lack specificity and overestimate the actual PTH (1–84) values because of cross-reactivity with PTH containing amino acids 7–84 (PTH 7–84) [37]. The artificially elevated iPTH levels may potentially hamper intraoperative evaluation of resection sufficiency leading to further surgical exploration that is otherwise unwarranted [37]. This problem of cross-reactivity with PTH (7–84) has prompted the development of a third generation of assays, which use antibodies targeted against an epitope containing the proximal 4–6 amino acids at the N-terminal [37]. While some authors propose that third-generation assays provide superior intraoperative data, it is not clear whether the implementation of these assays will translate into a reduction in failed parathyroid surgeries, decreased hypoparathyroidism, or decreased operating times [37]. It is unknown whether the clinical utility will ultimately exceed the longer incubation times and elevated costs associated with the newer assays.
Classical Actions of PTH
In general, continuous infusion of PTH causes calcium levels to rise until eventually reaching a plateau after about 4 h [27] (Figs. 4.6 and 4.7). In juxtaposition, the phosphate concentration declines relatively rapidly and reaches a plateau within 2 h [27]. This occurs because PTH increases the calcium and phosphate absorption from the bone and decreases the excretion of calcium by the kidneys. The PTH-induced decline in phosphate, then, is a consequence of the rapid excretion of phosphate, relative to the rate of phosphate reabsorption in bone [27].
Fig. 4.6
Approximate changes in calcium concentrations during the first 5 h of parathyroid hormone infusion at a moderate rate. From: Hall J. Parathyroid hormone, calcitonin, calcium and phosphate metabolism, vitamin D, bone and teeth, in Guyton and Hall textbook of medical physiology. Chapter 79. 11ed. Philadelphia: Saunders-Elsevier: 2011. Philadelphia 955–972
Fig. 4.7
Summary of parathyroid hormone (PTH) actions on bone, kidneys, and intestine in response to decreased extracellular calcium concentrations. CaSR, calcium-sensing receptor. From: Hall J. Parathyroid hormone, calcitonin, calcium and phosphate metabolism, vitamin D, bone and teeth, in Guyton and Hall textbook of medical physiology. Chapter 79. 11ed. Philadelphia: Saunders-Elsevier: 2011. Philadelphia 955–972
Bone
PTH acts on bone, containing upwards of 99 % of total body calcium stores, to release calcium in two stages [38]. The first phase, which occurs within minutes and rises steadily over hours, involves the release of calcium and phosphate stores from pre-existing bone cells. The bone is separated from the extracellular fluid by a membrane of interconnected osteoblasts and osteocytes referred to as the osteocytic membrane [27]. The osteocytic membrane contains a small amount of interim fluid, called bony fluid, whose calcium concentration is dictated by osteocytic pumps [26]. PTH binds to osteocyte receptors and increases the permeability of the bone fluid side of the osteocytic membrane, which causes the calcium concentration to increase in the bony fluid. The calcium ions that diffuse into the membrane cells from the bone fluid activate the calcium pump, which results in the rapid removal of calcium phosphate salts from the amorphous bone crystals that lie near the cells [27].
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