Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 206–212
ORIGINAL ARTICLE
Parathyroid hormone-related peptide plasma concentrations in patients on hemodialysis
ANDERS NORDHOLM1, MARIANNE RIX1, KLAUS OLGAARD1 & EWA LEWIN1,2 1University
of Copenhagen, Nephrological Department P, Rigshospitalet and 2University of Copenhagen, Nephrological Department B, Herlev Hospital, Copenhagen, Denmark
Abstract Background. Uremic patients develop hyperplasia of the parathyroid glands due to disturbances in the mineral metabolism. The hyperplastic parathyroids are associated with significant expression of parathyroid hormone (PTH)related peptide (PTHrP). PTHrP has been shown to have an autocrine/paracrine function in the parathyroids, but it is still uncertain if PTHrP is a secretory product of the gland and thereby possess endocrine actions. In cells of severe adenomatous secondary hyperparathyroidism PTHrP and PTH have been found to be co-localized in the same secretory granules. PTH and PTHrP act through the same receptor, the PTH1R, and it has been shown experimentally that PTHrP enhances the PTH secretory response to hypocalcemia, indicating a link between the two hormones. Methods. Together with a number of parameters involved in mineral homeostasis plasma PTHrP was measured before hemodialysis in 90 patients and in 15 healthy subjects. Plasma PTH was determined in order to examine the possible relationship between the two peptides. Results. In hemodialysis patients mean plasma PTHrP, 4.2 ⫾ 2.1, was significantly lower than that of healthy subjects, 8.3 ⫾ 1.1 pmol/L, p ⬍ 0.0001. No relationship was found between plasma PTHrP and PTH in hemodialysis patients. Gender, PTX, specific treatments and diagnoses had no impact on PTHrP concentrations. Conclusion. Thus PTHrP is measurable in hemodialysis patients, but its secretion might not be part of a regulated mineral homeostatic process and may not derive from the uremic hyperplastic parathyroid glands. Key Words: PTHrP, parathyroid gland, hyperplasia, secondary hyperparathyroidism, uremia, hemodialysis
Introduction It is known that chronic kidney disease (CKD) is associated with considerable morbidity and mortality related to disturbed mineral metabolism [1,2], and that even in early CKD the majority of the patients are suffering from secondary hyperparathyroidism (2HPT) [3]. In uremic patients, parathyroid growth takes place in response to chronic renal failure and progresses through several stages from diffuse hyperplasia to nodular hyperplasia and to formation of large adenomas [4–8]. Detailed histochemical and immunochemical studies indicate similarity in gene expression between the cells in each nodule, but differences between nodules [6,9]. The enlargement of the parathyroid glands in uremia can reach a magnitude of more than 1000-fold as compared to that of normal glands, thereby contributing to the severely increased secretion of PTH [10].
Parathyroid hormone related peptide (PTHrP) is a protein with sequential homology to parathyroid hormone (PTH) in its amino-terminal domain [10,11]. PTHrP was discovered in 1987 as a humoral factor responsible for humoral hypercalcemia of malignancy [12]. Many studies have focused on the physiological functions of PTHrP [13–17]. The PTHrP gene is expressed in essentially every tissue and organ at some point during fetal development or in adult life [14,18,19]. PTHrP influences proliferation and differentiation of a variety of cell types including bone cells, chondrocytes and keratinocytes [13]. In lactating organisms PTHrP is produced by mammary glands and contributes to calcium release from bone in order to supply the mineral for milk production. Mammary PTHrP is directly regulated by the calcium sensing receptor (CaR) [20]. PTHrP is expressed in the parathyroids
Correspondence: Anders Nordholm, MD, University of Copenhagen, Nephrological Department P 2132, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel: ⫹ 45 3545 2130. Fax: ⫹ 45 3545 2672. E-mail: [email protected] (Received 16 August 2013 ; accepted 15 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.876656
Plasma PTHrP in hemodialysis patients from fetal to adult life in different species [21–23]. PTHrP is expressed in normal human, bovine and rat parathyroid tissue [9,19,24,25] and an abnormal expression of PTHrP mRNA and protein has been demonstrated in human parathyroid adenomas, hyperplasia and carcinomas [19,26,27]. PTH and PTHrP are shown to be co-localized in the same secretory granules and secreted simultaneously from parathyroid adenomas [28]. In human parathyroid hyperplasia secondary to chronic renal failure a majority of the glands has been shown positive for PTHrP staining by immunohistochemistry, both in the diffuse and nodular type of hyperplasia. Furthermore, a negative correlation between parathyroid cell proliferation and PTHrP expression was found [9]. Parathyroid oxyphile cells are believed to be derived from parathyroid chief cells. Oxyphile cells have been shown to express high amount of PTHrP and the number of oxyphile cells are increased in parathyroid glands of CKD patients [29]. PTHrP exhibits biological similarities to transforming growth factor-ß and may have effects on cell growth or differentiation [13]. Previous results from our lab have shown a stimulatory effect of PTHrP on the secretion of PTH under hypocalcemic condition, supporting the possibility of an autocrine/paracrine function of PTHrP in the parathyroids [10]. In the fetus, PTHrP is the primary secretory protein in the parathyroid glands and plays a major role in the development of cartilage and bone [30]. However, the physiological role of PTHrP in the parathyroids still remains to be established. In healthy adult non-pregnant human subjects, the concentration of circulating PTHrP in blood is low [31], while the concentration is significantly elevated in some cases of humoral hypercalcemia of malignancy, e.g. in lung cancer [9,12,19,26,28]. PTHrP as well as PTH act through the same PTH/PTHrP receptor (PTH1R), a common G-protein linked receptor [15,32,33], which is widely distributed in the body, including in cells with relation to the mineral metabolism [13,32]. Thus, as a significant expression of PTHrP has been well documented in human uremic hyperplastic parathyroid glands, it is of relevance to examine whether the peptide is present at elevated concentrations in the circulation of uremic patients with secondary hyperparathyroidism. If that is the case, PTHrP might potentially contribute with some PTH-like activity and might be part of the severe disturbances in Chronic Kidney Disease Mineral and Bone Disorder (CKD-MBD). This has not been systematically examined before in a hemodialysis population.
Methods Study design The present investigation is a cross-sectional study, including 90 uremic patients on chronic hemodialysis
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(HD) at Nephrological Department P, Rigshospitalet, Denmark. All patients on HD were asked for permission to obtain an extra blood sample for measurement of PTHrP, beside their standard measurements of routine parameters. Fifteen healthy subjects were included to determine if PTHrP were measurable under normal conditions, all were medical students, who were asked randomly for participation. None of the participants were pregnant or lactating. Permission to perform the study was obtained from the Ethical Committee at the Capital Region, H-2-2012-133, in Denmark. The project was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000. Blood sampling Blood samples were collected from the hemodialysis patients prior to initiation of HD. Samples for PTHrP were drawn in two 5 mL EDTA tubes on ice, centrifuged immediately, and stored at ⫺20°C until analysis. Additional blood samples were obtained simultaneously for hemoglobin, ionized calcium (Ca2⫹), total calcium, phosphorous, alkaline phosphatase, magnesium, 25(OH)D, creatinine and PTH, all analyzed at the Department of Clinical Biochemistry, Rigshospitalet. Plasma PTH was measured by the intact PTH electrochemiluminescence immunoassay (ECLIA; Roche PTH), measuring intact PTH by the use of a biotinylated monoclonal antibody, which reacts with amino acids 26–32, and a capture rutheniumcomplexed monoclonal antibody, which reacts with amino acids 55–64. The determinations were performed on Roche Modular E 170®. The intra-assay variation is 4.1% and the inter-assay variation 5.8%. Blood samples from the group of normal subjects were handled in exactly the same way as the samples from the hemodialysis patients. All blood samples, both from HD patients and normal subjects were collected between mid-December 2012 and the end of January 2013. All were analyzed within two months.
PTHrP measurement Plasma PTHrP was analyzed with a sandwich ELISA technique by a commercial kit (catalogue number E-EL-H1478), from Elabscience Biotechnology Co., Ltd, China. This kit’s capture antibody aims at the 1-34 N-terminal region of PTHrP while a biotinylated detection antibody aims at the 39-106 region. The manufacture reports no cross reactivity with PTH. The minimal detectable concentration of PTHrP in our lab was 0.5 pmol/L. The intraassay variation was 10% in our lab and the interassay variation 7.2%. All samples were analyzed by duplicate determination.
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Statistical analyses
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Results
Table I. Characteristics of hemodialysis patients and normal subjects.
No. of patients Mean age in years (range) Male/female Partial PTX∗ Diagnosis Diabetes Mellitus Hypertension Chronic Uremia Chronic Glomerulonephritis Cystic kidney diseases Chronic Pyelonephritis Other Receiving treatment with: Calcimimetics 1,25OH2 Vitamin D analog Chole-/ergocalciferol Calcium antagonist Phosphate binder ∗PTX, Parathyroidectomy.
10
5
0
The basic characteristics of both the hemodialysis patients and the group of healthy subjects are shown in Table I. Both groups passed the normality test, and the Gaussian distribution of plasma PTHrP of the hemodialysis patients is presented in Figure 1. Plasma PTHrP were measurable in all samples, except one. Biochemical characteristics of all 90 hemodialysis patients and of all 15 healthy subjects are shown in Table II. The mean concentration of PTHrP in the group of hemodialysis patients was significantly lower (4.2 ⫾ 2.1 pmol/L) than that of the healthy subjects (8.3 ⫾ 1.1 pmol/L), p ⬍ 0.0001, as illustrated in Figure 2. Table III shows mean plasma PTHrP concentrations in relation to gender, PTX and treatment with calcimimetics, 1.25(OH)2 vitamin D analogs, chole/ ergocalciferol, calcium antagonists or phosphate binders. No significant differences were found between the groups.
Characteristics
15 No. of Patients
The analyses were performed using GraphPad Prism 4 and SPSS 19. The data are expressed as mean values ⫾ standard deviations (SD). As the data had Gaussian distribution, hemodialysis patients and normal subjects were compared to each other with Student’s t-test; p ⬍ 0.05 was considered significant. One-way ANOVA was used to compare mean PTHrP concentrations related to renal diagnosis. The relationship between plasma PTHrP and other variables were analyzed with no adjustments for potential confounders by using the squared Pearson’s correlation and linear regressions.
Normal subjects
Hemodialysis patients
15 27 (25–29) 9/6
90 65 (24–93) 58/32 4 25 17 16 12 5 8 7 16 63 12 22 72
0
1 2 3 4 5 6 7 8 9 10 11 12 PTHrP concentration intervals (pmol/L)
Figure 1. Gaussian distribution of plasma PTHrP in hemodialysis patients. Mean ⫽ 4.2 ⫾ 2.1 pmol/L; n ⫽ 90.
PTHrP concentrations in relation to groups of different renal diagnoses were examined, but no significant difference was found between the different groups of patients, p ⬎ 0.05. The relationship between PTHrP and age among the hemodialysis patients is shown in Figure 3. A significant, although week correlation was found between increasing plasma PTHrP and increasing age (p ⬍ 0.05, r2 ⫽ 0.05). The relationship between plasma PTHrP and all biochemical plasma concentrations, presented in Table II, were analyzed by linear regression and correlation, with no significant associations found (data not shown). Figure 4 shows the relationship between plasma PTHrP and PTH in all hemodialysis patients. No significant association was found, p ⬎ 0.05, r2 ⫽ 0.03. Including only the patients with hypocalcemia did not change the lack of association, p ⬎ 0.05, r ⫽ 0.04, n ⫽ 51. The 39 patients with ionized calcium concentrations above 1.18 mmol/L (lower limit at Rigshospitalet) were not included in this part. Thus no significant correlation was seen between the plasma concentrations of the two peptides in neither the group of all patients on hemodialysis nor in the group of patients, supposed to have stimulated PTH concentration. Adjusting for partial PTX and/or treatment with calcimimetics did not affect the lack of relationship (data not shown).
Discussion The aim of this study was to examine whether PTHrP was present and, in that case, present at elevated concentrations, in the circulation of hemodialysis patients with secondary hyperparathyroidism.
Plasma PTHrP in hemodialysis patients
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Table II. Biochemical characteristics of hemodialysis patients and normal subjects. Plasma concentrations are shown as mean ⫾ SD. Biochemical characteristics Plasma concentrations PTHrP pmol/L Range pmol/L PTH pmol/L Ca2⫹ mmol/L Total calcium mmol/L Phosphate mmol/L Alkaline phosphatase U/L Magnesium mmol/L 25(OH) Vitamin D∗∗∗ nmol/L Creatinine∗∗∗ μmol/L Hemoglobin mmol/L
Normal subjects
Hemodialysis patients
8.3 ⫾ 1.1 5.9–10.0 3.7 ⫾ 0.1 1.23 ⫾ 0.03 mmol/L ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗
4.2 ⫾ 2.1 0.0–12.2 32.6 ⫾ 25.6 1.16 ⫾ 0.08 2.19 ⫾ 0.56 1.69 ⫾ 0.55 95 ⫾ 62 0.89 ⫾ 0.26 36.1 ⫾ 23.0 696 ⫾ 234 7.3 ⫾ 0.8
Reference∗
1.6–6.9 1.18–1.32 2.15–2.51 0.71–1.23 35–105 0.71–0.94 ⬎ 50 60–105 8.3–10.5
mm/L mmoL mmol/L mmol/L
∗At Rigshospitalet; ∗∗Not analyzed in normal subjects; ∗∗∗Five and one patients respectively are excluded due to missing data.
Elevated PTHrP could potentially be derived from severely enlarged parathyroid glands, characteristic for the uremic condition [4,5]. As such a significant overexpression of PTHrP has previously been found in the uremic hyperplastic parathyroids where PTHrP was present together with PTH in the secretory granules [9,28]. Plasma PTHrP was in the present study measurable in all the patients on hemodialysis, except one. The present results, however do not support the notion of circulating PTHrP being derived especially from overactive uremic parathyroid glands, as the plasma concentrations of PTHrP were not increased in the uremic patients, and as no correlation between plasma PTHrP and plasma PTH was found. Even in hypocalcemia, when PTH secretion is stimulated [10], no correlation between the two peptides was found.
Table III. PTHrP mean concentrations related to gender, PTX and specific treatment.
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Plasma PTHrP (pmol/L)
PTHrP is widely expressed in human tissues and the concentrations found in both hemodialysis patients and normal subjects probably might derive from sources other than the parathyroids [13,17,18,21]. The exact role of PTHrP in the parathyroids has not yet been established and the significantly overexpressed parathyroid PTHrP in uremia is suggested to have an autocrine/paracrine role, however without necessarily being an endocrine secretory product of the gland in the present study. When interpreting the present results one has to take into consideration that the large expression of parathyroid PTHrP in uremia was found in previous studies on limited subgroups of uremic patients where the parathyroid glands were obtained due to very severe secondary hyperparathyroidism,
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Figure 2. Plasma PTHrP in normal subjects versus hemodialysis patients. Plasma PTHrP was significantly lower among hemodialysis patients compared to a group of normal subjects, p ⬍ 0.0001.
Gender Male Female PTX No Yes Calcimimetics No Yes 1,25OH Vitamin D analog No Yes Chole-/ergocalciferol No Yes Calcium antagonist No Yes Phosphate binder No Yes
N
Mean ⫾ SD pmol/L
p
58 32
4.3 ⫾ 2.2 4.1 ⫾ 2.0
ns
86 4
4.2 ⫾ 2.2 4.9 ⫾ 1.3
ns
74 16
4.2 ⫾ 2.1 4.3 ⫾ 2.4
ns
27 63
4.3 ⫾ 2.4 4.1 ⫾ 2.0
ns
78 12
4.3 ⫾ 2.2 3.8 ⫾ 1.9
ns
68 22
4.1 ⫾ 2.0 4.4 ⫾ 2.6
ns
18 72
4.4 ⫾ 1.7 4.1 ⫾ 2.2
ns
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Plasma PTHrP (pmol/L)
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0 0
25
50 Age (years)
75
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Figure 3. Plasma PTHrP versus age in hemodialysis patients. A weak correlation between plasma PTHrP and age was demonstrated. Plasma PTHrP increases with rising age (p ⬍ 0.05, n ⫽ 90).
demanding surgical removal of the glands [9], in contrast to the present cross-sectional study. Both hemodialysis patients and normal subjects had measurable plasma PTHrP, except in one patient. In normal subjects low concentrations of plasma PTHrP have previously been reported in studies including small number of subjects [31,34], except in lactating women, where the concentrations were enhanced [35,36]. The normal subjects in the present study had a mean of 8.3 ⫾ 1.1 pmol/L, which might be slightly higher than that reported in previous studies that mainly were focusing on conditions related to humoral hypercalcemia of malignancy, and where plasma PTHrP were measured by immunoradiometric assays [31,34]. In human subjects PTHrP is potentially present in three different isoforms of 139, 141, and 173 amino acids [37]. The present ELISA assay using N-terminal and mid-molecular antibodies should detect the different splice forms. The difference between PTHrP concentrations in the different studies might be related to the sensitivity of the different assays. Acceptable low inter- and intra-assay
Plasma PTHrP (pmol/L)
15
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0 0
25
50 75 100 Plasma PTH (pmol/L)
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Figure 4. Plasma PTHrP versus plasma PTH in hemodialysis patients. No significant correlations were found between plasma PTHrP and PTH in hemodialysis patients. This is illustrated for all hemodialysis patients (ns, n ⫽ 90).
variations and good reproducibility for the kit used was found in the present study. Plasma PTHrP in hemodialysis patients were slightly (p ⬍ 0.05) lower than in the normal subjects, indicating no extra significant contribution of the uremic state to the plasma concentrations and suggesting that the metabolism might be different in the two situations. The samples from both hemodialysis patients and normal subjects were treated similarly and stored for approximately the same time and therefore should not contribute to any difference. It should however be noted, that there was a major difference in the mean age between the hemodialysis patients (mean age 65 years) and the group of normal subjects (mean age 27 years). There were no noticeable difference in distribution of gender between the groups and the normal subjects were not considered as a ‘normal control group’, but just used as reference for measuring PTHrP concentrations in non-uremic subjects. It would of course be interesting in future studies to compare plasma PTHrP among hemodialysis patients with a larger group of comparable normal subjects. It is known that PTHrP is significantly elevated in the presence of humoral hypercalcemia of malignancy due to cancer [9,12,19,26,28,31]. Unfortunately, in the present study we did not have the possibility to include such positive control patients. No correlation between plasma PTHrP and any of the other measured biochemical markers showed significant values. Regarding 25OH vitamin D, one has to notice that the group of hemodialysis patients in this study were not 25OH vitamin D sufficient. Parathyroid cell expresses indeed 1α-hydroxylase activity and an effect of the parathyroid vitamin D autocrine system on PTH biosynthesis has been proposed. Whether local 1αhydroxylase activity and vitamin D status among CKD patients are of importance for PTHrP regulation is as yet not known. The recent paper of Ritter et al. [29] suggested the existence of increased expression of the 1α-hydroxylase in oxyphile parathyroid cells, which in other studies are expressing high amounts of PTHrP protein too [9,29]. In the present study no relationship was found between plasma PTHrP and PTH. The scatter plot derived from all the hemodialysis patients showed no significant link (Figure 4). Adjusting for PTX, specific treatment and renal diagnoses did not induce or reveal significant relationships between PTHrP and PTH, also when studying the relationship only in hypocalcemic hemodialysis patients. The interpretation of PTH concentrations in two subgroups of patients, partial PTX patients and patients treated with calcimimetics demands for special considerations. Patients treated with partial PTX have a potentially impaired parathyroid function as a consequence of the now diminished parathyroid mass.
Plasma PTHrP in hemodialysis patients Calcimimetic treatment alters the relationship between PTH and calcium, shifting the sigmoidal curve leftwards [38]. In addition, PTH concentrations are difficult to interpret in calcimimetic-treated patients due to specific pharmacokinetics of the compound, as the maximal effect on PTH suppression occurs approximately 3 h after ingestion, where after PTH rises again. Nevertheless exclusion of patients treated with PTX and calcimimetics did not have an impact on the lack of relationship between PTHrP and PTH or on the concentrations of PTHrP. In uremia, female gender is associated with larger hyperplastic parathyroid glands [39]. In the present study however gender did not affect the plasma PTHrP. We have previously shown experimentally in normal and uremic rats, that exogenous PTHrP stimulated PTH secretion during hypocalcemic conditions [10,40]. In the present study it was however not possible to examine for such a connection just by measuring the circulating concentrations of the two peptides. This does however not exclude the possibility of an autocrine/paracrine action of PTHrP on PTH secretion at the glandular level. Furthermore, in our previous experimental studies N-terminal PTHrP was used as a surrogate for PTH as activator of the common PTH/PTHrP receptor. The enhanced stimulated PTH secretion might represent the auto-stimulatory effect of PTH under condition of hypocalcemia [10,40]. Parathyroid PTHrP has been proposed to be associated negatively to parathyroid cell proliferation, thus suppressing parathyroid growth [9]. In a recent study, Ritter et al. [29] demonstrated a huge content of oxyphile cells in the glands from uremic patients. Chief cells are the major cell type of the parathyroid glands in healthy young subjects, while the oxyphile cells appear with increasing age and increases further dramatically in number in patients with CKD. This oxyphile cell type is characterized by high expression of PTH and PTHrP, making future studies on the role of PTHrP in uremic parathyroids very relevant.
Conclusion Plasma PTHrP concentrations were clearly measurable in uremic patients on chronic hemodialysis and corresponded positively to age. Plasma PTHrP did not correlate to plasma PTH concentration neither in all patients on hemodialysis nor in those with hypocalcemia and as such stimulated PTH secretion. This might indicate that the enhanced expression in uremia of PTHrP in the parathyroid glands in secondary hyperparathyroidism, might not directly be transferred to the circulation, but would theoretically be of importance for the autocrine/paracrine functions within the enlarged parathyroid glands.
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Acknowledgements The authors thank technician Kirsten Bang for her excellent contribution.
Funding sources No external funding sources were received. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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abnormal human parathyroids. J Endocrinol 1991;129: 431–8. Matsushita H, Usui M, Hara M, Shishiba Y, Nakazawa H, Honda K, Torigoe K, Kohno K, Kurimoto M. Co-secretion of parathyroid hormone and parathyroidhormone-related protein via a regulated pathway in human parathyroid adenoma cells. Am J Pathol 1997;150:861–71. Ritter CS, Haughey BH, Miller B, Brown AJ. Differential gene expression by oxyphil and chief cells of human parathyroid glands. J Clin Endocrinol Metab 2012;97: E1499–505. Kronenberg HM. PTHrP and skeletal development. Ann NY Acad Sci 2006;1068:1–13. de Miguel F, Motellon JL, Hurtado J, Jimenez FJ, Esbrit P. Comparison of two immunoradiometric assays for parathyroid hormone-related protein in the evaluation of cancer patients with and without hypercalcemia. Clin Chim Acta 1998;277:171–80. Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH, Vasavada RC, Weir EC, Broadus AE, Stewart AF. Defining the roles of parathyroid hormonerelated protein in normal physiology. Physiol Rev 1996; 76:127–73. Mangin M, Webb AC, Dreyer BE, Posillico JT, Ikeda K, Weir EC, Stewart AF, Bander NH, Milstone L, Barton DE. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy. Proc Natl Acad Sci USA 1988;85:597–601. Takahashi S, Hakuta M, Aiba K, Ito Y, Horikoshi N, Miura M, Hatake K, Ogata E. Elevation of circulating plasma cytokines in cancer patients with high plasma parathyroid hormone-related protein levels. Endocr Relat Cancer 2003;10:403–7. Grill V, Hillary J, Ho PM, Law FM, MacIsaac RJ, MacIsaac IA, Moseley JM, Martin TJ. Parathyroid hormonerelated protein: a possible endocrine function in lactation. Clin Endocrinol (Oxf) 1992;37:405–10. Bucht E, Rong H, Bremme K, Granberg B, Rian E, Torring O. Midmolecular parathyroid hormone-related peptide in serum during pregnancy, lactation and in umbilical cord blood. Eur J Endocrinol 1995;132:438–43. Power DM, Ingleton PM, Flanagan J, Canario AV, Danks J, Elgar G, Clark MS. Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes. Gene 2000;250: 67–76. de Francisco AL, Izquierdo M, Cunningham J, Pinera C, Palomar R, Fresnedo GF, Amado JA, Unzueta MG, Arias M. Calcium-mediated parathyroid hormone release changes in patients treated with the calcimimetic agent cinacalcet. Nephrol Dial Transplant 2008;23:2895–901. Basile C, Lomonte C, Vernaglione L, Casucci F, Chimienti D, Bruno A, Cocola S, Verrelli EA, Cazzato F. A high body mass index and female gender are associated with an increased risk of nodular hyperplasia of parathyroid glands in chronic uraemia. Nephrol Dial Transplant 2006;21: 968–74. Lewin E, Garfia B, Almaden Y, Rodriguez M, Olgaard K. Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats. Kidney Int 2003;64:63–70.
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ORIGINAL ARTICLE
Parathyroid hormone-related peptide plasma concentrations in patients on hemodialysis
ANDERS NORDHOLM1, MARIANNE RIX1, KLAUS OLGAARD1 & EWA LEWIN1,2 1University
of Copenhagen, Nephrological Department P, Rigshospitalet and 2University of Copenhagen, Nephrological Department B, Herlev Hospital, Copenhagen, Denmark
Abstract Background. Uremic patients develop hyperplasia of the parathyroid glands due to disturbances in the mineral metabolism. The hyperplastic parathyroids are associated with significant expression of parathyroid hormone (PTH)related peptide (PTHrP). PTHrP has been shown to have an autocrine/paracrine function in the parathyroids, but it is still uncertain if PTHrP is a secretory product of the gland and thereby possess endocrine actions. In cells of severe adenomatous secondary hyperparathyroidism PTHrP and PTH have been found to be co-localized in the same secretory granules. PTH and PTHrP act through the same receptor, the PTH1R, and it has been shown experimentally that PTHrP enhances the PTH secretory response to hypocalcemia, indicating a link between the two hormones. Methods. Together with a number of parameters involved in mineral homeostasis plasma PTHrP was measured before hemodialysis in 90 patients and in 15 healthy subjects. Plasma PTH was determined in order to examine the possible relationship between the two peptides. Results. In hemodialysis patients mean plasma PTHrP, 4.2 ⫾ 2.1, was significantly lower than that of healthy subjects, 8.3 ⫾ 1.1 pmol/L, p ⬍ 0.0001. No relationship was found between plasma PTHrP and PTH in hemodialysis patients. Gender, PTX, specific treatments and diagnoses had no impact on PTHrP concentrations. Conclusion. Thus PTHrP is measurable in hemodialysis patients, but its secretion might not be part of a regulated mineral homeostatic process and may not derive from the uremic hyperplastic parathyroid glands. Key Words: PTHrP, parathyroid gland, hyperplasia, secondary hyperparathyroidism, uremia, hemodialysis
Introduction It is known that chronic kidney disease (CKD) is associated with considerable morbidity and mortality related to disturbed mineral metabolism [1,2], and that even in early CKD the majority of the patients are suffering from secondary hyperparathyroidism (2HPT) [3]. In uremic patients, parathyroid growth takes place in response to chronic renal failure and progresses through several stages from diffuse hyperplasia to nodular hyperplasia and to formation of large adenomas [4–8]. Detailed histochemical and immunochemical studies indicate similarity in gene expression between the cells in each nodule, but differences between nodules [6,9]. The enlargement of the parathyroid glands in uremia can reach a magnitude of more than 1000-fold as compared to that of normal glands, thereby contributing to the severely increased secretion of PTH [10].
Parathyroid hormone related peptide (PTHrP) is a protein with sequential homology to parathyroid hormone (PTH) in its amino-terminal domain [10,11]. PTHrP was discovered in 1987 as a humoral factor responsible for humoral hypercalcemia of malignancy [12]. Many studies have focused on the physiological functions of PTHrP [13–17]. The PTHrP gene is expressed in essentially every tissue and organ at some point during fetal development or in adult life [14,18,19]. PTHrP influences proliferation and differentiation of a variety of cell types including bone cells, chondrocytes and keratinocytes [13]. In lactating organisms PTHrP is produced by mammary glands and contributes to calcium release from bone in order to supply the mineral for milk production. Mammary PTHrP is directly regulated by the calcium sensing receptor (CaR) [20]. PTHrP is expressed in the parathyroids
Correspondence: Anders Nordholm, MD, University of Copenhagen, Nephrological Department P 2132, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel: ⫹ 45 3545 2130. Fax: ⫹ 45 3545 2672. E-mail: [email protected] (Received 16 August 2013 ; accepted 15 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.876656
Plasma PTHrP in hemodialysis patients from fetal to adult life in different species [21–23]. PTHrP is expressed in normal human, bovine and rat parathyroid tissue [9,19,24,25] and an abnormal expression of PTHrP mRNA and protein has been demonstrated in human parathyroid adenomas, hyperplasia and carcinomas [19,26,27]. PTH and PTHrP are shown to be co-localized in the same secretory granules and secreted simultaneously from parathyroid adenomas [28]. In human parathyroid hyperplasia secondary to chronic renal failure a majority of the glands has been shown positive for PTHrP staining by immunohistochemistry, both in the diffuse and nodular type of hyperplasia. Furthermore, a negative correlation between parathyroid cell proliferation and PTHrP expression was found [9]. Parathyroid oxyphile cells are believed to be derived from parathyroid chief cells. Oxyphile cells have been shown to express high amount of PTHrP and the number of oxyphile cells are increased in parathyroid glands of CKD patients [29]. PTHrP exhibits biological similarities to transforming growth factor-ß and may have effects on cell growth or differentiation [13]. Previous results from our lab have shown a stimulatory effect of PTHrP on the secretion of PTH under hypocalcemic condition, supporting the possibility of an autocrine/paracrine function of PTHrP in the parathyroids [10]. In the fetus, PTHrP is the primary secretory protein in the parathyroid glands and plays a major role in the development of cartilage and bone [30]. However, the physiological role of PTHrP in the parathyroids still remains to be established. In healthy adult non-pregnant human subjects, the concentration of circulating PTHrP in blood is low [31], while the concentration is significantly elevated in some cases of humoral hypercalcemia of malignancy, e.g. in lung cancer [9,12,19,26,28]. PTHrP as well as PTH act through the same PTH/PTHrP receptor (PTH1R), a common G-protein linked receptor [15,32,33], which is widely distributed in the body, including in cells with relation to the mineral metabolism [13,32]. Thus, as a significant expression of PTHrP has been well documented in human uremic hyperplastic parathyroid glands, it is of relevance to examine whether the peptide is present at elevated concentrations in the circulation of uremic patients with secondary hyperparathyroidism. If that is the case, PTHrP might potentially contribute with some PTH-like activity and might be part of the severe disturbances in Chronic Kidney Disease Mineral and Bone Disorder (CKD-MBD). This has not been systematically examined before in a hemodialysis population.
Methods Study design The present investigation is a cross-sectional study, including 90 uremic patients on chronic hemodialysis
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(HD) at Nephrological Department P, Rigshospitalet, Denmark. All patients on HD were asked for permission to obtain an extra blood sample for measurement of PTHrP, beside their standard measurements of routine parameters. Fifteen healthy subjects were included to determine if PTHrP were measurable under normal conditions, all were medical students, who were asked randomly for participation. None of the participants were pregnant or lactating. Permission to perform the study was obtained from the Ethical Committee at the Capital Region, H-2-2012-133, in Denmark. The project was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000. Blood sampling Blood samples were collected from the hemodialysis patients prior to initiation of HD. Samples for PTHrP were drawn in two 5 mL EDTA tubes on ice, centrifuged immediately, and stored at ⫺20°C until analysis. Additional blood samples were obtained simultaneously for hemoglobin, ionized calcium (Ca2⫹), total calcium, phosphorous, alkaline phosphatase, magnesium, 25(OH)D, creatinine and PTH, all analyzed at the Department of Clinical Biochemistry, Rigshospitalet. Plasma PTH was measured by the intact PTH electrochemiluminescence immunoassay (ECLIA; Roche PTH), measuring intact PTH by the use of a biotinylated monoclonal antibody, which reacts with amino acids 26–32, and a capture rutheniumcomplexed monoclonal antibody, which reacts with amino acids 55–64. The determinations were performed on Roche Modular E 170®. The intra-assay variation is 4.1% and the inter-assay variation 5.8%. Blood samples from the group of normal subjects were handled in exactly the same way as the samples from the hemodialysis patients. All blood samples, both from HD patients and normal subjects were collected between mid-December 2012 and the end of January 2013. All were analyzed within two months.
PTHrP measurement Plasma PTHrP was analyzed with a sandwich ELISA technique by a commercial kit (catalogue number E-EL-H1478), from Elabscience Biotechnology Co., Ltd, China. This kit’s capture antibody aims at the 1-34 N-terminal region of PTHrP while a biotinylated detection antibody aims at the 39-106 region. The manufacture reports no cross reactivity with PTH. The minimal detectable concentration of PTHrP in our lab was 0.5 pmol/L. The intraassay variation was 10% in our lab and the interassay variation 7.2%. All samples were analyzed by duplicate determination.
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Statistical analyses
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Results
Table I. Characteristics of hemodialysis patients and normal subjects.
No. of patients Mean age in years (range) Male/female Partial PTX∗ Diagnosis Diabetes Mellitus Hypertension Chronic Uremia Chronic Glomerulonephritis Cystic kidney diseases Chronic Pyelonephritis Other Receiving treatment with: Calcimimetics 1,25OH2 Vitamin D analog Chole-/ergocalciferol Calcium antagonist Phosphate binder ∗PTX, Parathyroidectomy.
10
5
0
The basic characteristics of both the hemodialysis patients and the group of healthy subjects are shown in Table I. Both groups passed the normality test, and the Gaussian distribution of plasma PTHrP of the hemodialysis patients is presented in Figure 1. Plasma PTHrP were measurable in all samples, except one. Biochemical characteristics of all 90 hemodialysis patients and of all 15 healthy subjects are shown in Table II. The mean concentration of PTHrP in the group of hemodialysis patients was significantly lower (4.2 ⫾ 2.1 pmol/L) than that of the healthy subjects (8.3 ⫾ 1.1 pmol/L), p ⬍ 0.0001, as illustrated in Figure 2. Table III shows mean plasma PTHrP concentrations in relation to gender, PTX and treatment with calcimimetics, 1.25(OH)2 vitamin D analogs, chole/ ergocalciferol, calcium antagonists or phosphate binders. No significant differences were found between the groups.
Characteristics
15 No. of Patients
The analyses were performed using GraphPad Prism 4 and SPSS 19. The data are expressed as mean values ⫾ standard deviations (SD). As the data had Gaussian distribution, hemodialysis patients and normal subjects were compared to each other with Student’s t-test; p ⬍ 0.05 was considered significant. One-way ANOVA was used to compare mean PTHrP concentrations related to renal diagnosis. The relationship between plasma PTHrP and other variables were analyzed with no adjustments for potential confounders by using the squared Pearson’s correlation and linear regressions.
Normal subjects
Hemodialysis patients
15 27 (25–29) 9/6
90 65 (24–93) 58/32 4 25 17 16 12 5 8 7 16 63 12 22 72
0
1 2 3 4 5 6 7 8 9 10 11 12 PTHrP concentration intervals (pmol/L)
Figure 1. Gaussian distribution of plasma PTHrP in hemodialysis patients. Mean ⫽ 4.2 ⫾ 2.1 pmol/L; n ⫽ 90.
PTHrP concentrations in relation to groups of different renal diagnoses were examined, but no significant difference was found between the different groups of patients, p ⬎ 0.05. The relationship between PTHrP and age among the hemodialysis patients is shown in Figure 3. A significant, although week correlation was found between increasing plasma PTHrP and increasing age (p ⬍ 0.05, r2 ⫽ 0.05). The relationship between plasma PTHrP and all biochemical plasma concentrations, presented in Table II, were analyzed by linear regression and correlation, with no significant associations found (data not shown). Figure 4 shows the relationship between plasma PTHrP and PTH in all hemodialysis patients. No significant association was found, p ⬎ 0.05, r2 ⫽ 0.03. Including only the patients with hypocalcemia did not change the lack of association, p ⬎ 0.05, r ⫽ 0.04, n ⫽ 51. The 39 patients with ionized calcium concentrations above 1.18 mmol/L (lower limit at Rigshospitalet) were not included in this part. Thus no significant correlation was seen between the plasma concentrations of the two peptides in neither the group of all patients on hemodialysis nor in the group of patients, supposed to have stimulated PTH concentration. Adjusting for partial PTX and/or treatment with calcimimetics did not affect the lack of relationship (data not shown).
Discussion The aim of this study was to examine whether PTHrP was present and, in that case, present at elevated concentrations, in the circulation of hemodialysis patients with secondary hyperparathyroidism.
Plasma PTHrP in hemodialysis patients
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Table II. Biochemical characteristics of hemodialysis patients and normal subjects. Plasma concentrations are shown as mean ⫾ SD. Biochemical characteristics Plasma concentrations PTHrP pmol/L Range pmol/L PTH pmol/L Ca2⫹ mmol/L Total calcium mmol/L Phosphate mmol/L Alkaline phosphatase U/L Magnesium mmol/L 25(OH) Vitamin D∗∗∗ nmol/L Creatinine∗∗∗ μmol/L Hemoglobin mmol/L
Normal subjects
Hemodialysis patients
8.3 ⫾ 1.1 5.9–10.0 3.7 ⫾ 0.1 1.23 ⫾ 0.03 mmol/L ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗
4.2 ⫾ 2.1 0.0–12.2 32.6 ⫾ 25.6 1.16 ⫾ 0.08 2.19 ⫾ 0.56 1.69 ⫾ 0.55 95 ⫾ 62 0.89 ⫾ 0.26 36.1 ⫾ 23.0 696 ⫾ 234 7.3 ⫾ 0.8
Reference∗
1.6–6.9 1.18–1.32 2.15–2.51 0.71–1.23 35–105 0.71–0.94 ⬎ 50 60–105 8.3–10.5
mm/L mmoL mmol/L mmol/L
∗At Rigshospitalet; ∗∗Not analyzed in normal subjects; ∗∗∗Five and one patients respectively are excluded due to missing data.
Elevated PTHrP could potentially be derived from severely enlarged parathyroid glands, characteristic for the uremic condition [4,5]. As such a significant overexpression of PTHrP has previously been found in the uremic hyperplastic parathyroids where PTHrP was present together with PTH in the secretory granules [9,28]. Plasma PTHrP was in the present study measurable in all the patients on hemodialysis, except one. The present results, however do not support the notion of circulating PTHrP being derived especially from overactive uremic parathyroid glands, as the plasma concentrations of PTHrP were not increased in the uremic patients, and as no correlation between plasma PTHrP and plasma PTH was found. Even in hypocalcemia, when PTH secretion is stimulated [10], no correlation between the two peptides was found.
Table III. PTHrP mean concentrations related to gender, PTX and specific treatment.
15
Plasma PTHrP (pmol/L)
PTHrP is widely expressed in human tissues and the concentrations found in both hemodialysis patients and normal subjects probably might derive from sources other than the parathyroids [13,17,18,21]. The exact role of PTHrP in the parathyroids has not yet been established and the significantly overexpressed parathyroid PTHrP in uremia is suggested to have an autocrine/paracrine role, however without necessarily being an endocrine secretory product of the gland in the present study. When interpreting the present results one has to take into consideration that the large expression of parathyroid PTHrP in uremia was found in previous studies on limited subgroups of uremic patients where the parathyroid glands were obtained due to very severe secondary hyperparathyroidism,
10
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s
s
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Figure 2. Plasma PTHrP in normal subjects versus hemodialysis patients. Plasma PTHrP was significantly lower among hemodialysis patients compared to a group of normal subjects, p ⬍ 0.0001.
Gender Male Female PTX No Yes Calcimimetics No Yes 1,25OH Vitamin D analog No Yes Chole-/ergocalciferol No Yes Calcium antagonist No Yes Phosphate binder No Yes
N
Mean ⫾ SD pmol/L
p
58 32
4.3 ⫾ 2.2 4.1 ⫾ 2.0
ns
86 4
4.2 ⫾ 2.2 4.9 ⫾ 1.3
ns
74 16
4.2 ⫾ 2.1 4.3 ⫾ 2.4
ns
27 63
4.3 ⫾ 2.4 4.1 ⫾ 2.0
ns
78 12
4.3 ⫾ 2.2 3.8 ⫾ 1.9
ns
68 22
4.1 ⫾ 2.0 4.4 ⫾ 2.6
ns
18 72
4.4 ⫾ 1.7 4.1 ⫾ 2.2
ns
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Plasma PTHrP (pmol/L)
15
10
5
0 0
25
50 Age (years)
75
100
Figure 3. Plasma PTHrP versus age in hemodialysis patients. A weak correlation between plasma PTHrP and age was demonstrated. Plasma PTHrP increases with rising age (p ⬍ 0.05, n ⫽ 90).
demanding surgical removal of the glands [9], in contrast to the present cross-sectional study. Both hemodialysis patients and normal subjects had measurable plasma PTHrP, except in one patient. In normal subjects low concentrations of plasma PTHrP have previously been reported in studies including small number of subjects [31,34], except in lactating women, where the concentrations were enhanced [35,36]. The normal subjects in the present study had a mean of 8.3 ⫾ 1.1 pmol/L, which might be slightly higher than that reported in previous studies that mainly were focusing on conditions related to humoral hypercalcemia of malignancy, and where plasma PTHrP were measured by immunoradiometric assays [31,34]. In human subjects PTHrP is potentially present in three different isoforms of 139, 141, and 173 amino acids [37]. The present ELISA assay using N-terminal and mid-molecular antibodies should detect the different splice forms. The difference between PTHrP concentrations in the different studies might be related to the sensitivity of the different assays. Acceptable low inter- and intra-assay
Plasma PTHrP (pmol/L)
15
10
5
0 0
25
50 75 100 Plasma PTH (pmol/L)
125
150
Figure 4. Plasma PTHrP versus plasma PTH in hemodialysis patients. No significant correlations were found between plasma PTHrP and PTH in hemodialysis patients. This is illustrated for all hemodialysis patients (ns, n ⫽ 90).
variations and good reproducibility for the kit used was found in the present study. Plasma PTHrP in hemodialysis patients were slightly (p ⬍ 0.05) lower than in the normal subjects, indicating no extra significant contribution of the uremic state to the plasma concentrations and suggesting that the metabolism might be different in the two situations. The samples from both hemodialysis patients and normal subjects were treated similarly and stored for approximately the same time and therefore should not contribute to any difference. It should however be noted, that there was a major difference in the mean age between the hemodialysis patients (mean age 65 years) and the group of normal subjects (mean age 27 years). There were no noticeable difference in distribution of gender between the groups and the normal subjects were not considered as a ‘normal control group’, but just used as reference for measuring PTHrP concentrations in non-uremic subjects. It would of course be interesting in future studies to compare plasma PTHrP among hemodialysis patients with a larger group of comparable normal subjects. It is known that PTHrP is significantly elevated in the presence of humoral hypercalcemia of malignancy due to cancer [9,12,19,26,28,31]. Unfortunately, in the present study we did not have the possibility to include such positive control patients. No correlation between plasma PTHrP and any of the other measured biochemical markers showed significant values. Regarding 25OH vitamin D, one has to notice that the group of hemodialysis patients in this study were not 25OH vitamin D sufficient. Parathyroid cell expresses indeed 1α-hydroxylase activity and an effect of the parathyroid vitamin D autocrine system on PTH biosynthesis has been proposed. Whether local 1αhydroxylase activity and vitamin D status among CKD patients are of importance for PTHrP regulation is as yet not known. The recent paper of Ritter et al. [29] suggested the existence of increased expression of the 1α-hydroxylase in oxyphile parathyroid cells, which in other studies are expressing high amounts of PTHrP protein too [9,29]. In the present study no relationship was found between plasma PTHrP and PTH. The scatter plot derived from all the hemodialysis patients showed no significant link (Figure 4). Adjusting for PTX, specific treatment and renal diagnoses did not induce or reveal significant relationships between PTHrP and PTH, also when studying the relationship only in hypocalcemic hemodialysis patients. The interpretation of PTH concentrations in two subgroups of patients, partial PTX patients and patients treated with calcimimetics demands for special considerations. Patients treated with partial PTX have a potentially impaired parathyroid function as a consequence of the now diminished parathyroid mass.
Plasma PTHrP in hemodialysis patients Calcimimetic treatment alters the relationship between PTH and calcium, shifting the sigmoidal curve leftwards [38]. In addition, PTH concentrations are difficult to interpret in calcimimetic-treated patients due to specific pharmacokinetics of the compound, as the maximal effect on PTH suppression occurs approximately 3 h after ingestion, where after PTH rises again. Nevertheless exclusion of patients treated with PTX and calcimimetics did not have an impact on the lack of relationship between PTHrP and PTH or on the concentrations of PTHrP. In uremia, female gender is associated with larger hyperplastic parathyroid glands [39]. In the present study however gender did not affect the plasma PTHrP. We have previously shown experimentally in normal and uremic rats, that exogenous PTHrP stimulated PTH secretion during hypocalcemic conditions [10,40]. In the present study it was however not possible to examine for such a connection just by measuring the circulating concentrations of the two peptides. This does however not exclude the possibility of an autocrine/paracrine action of PTHrP on PTH secretion at the glandular level. Furthermore, in our previous experimental studies N-terminal PTHrP was used as a surrogate for PTH as activator of the common PTH/PTHrP receptor. The enhanced stimulated PTH secretion might represent the auto-stimulatory effect of PTH under condition of hypocalcemia [10,40]. Parathyroid PTHrP has been proposed to be associated negatively to parathyroid cell proliferation, thus suppressing parathyroid growth [9]. In a recent study, Ritter et al. [29] demonstrated a huge content of oxyphile cells in the glands from uremic patients. Chief cells are the major cell type of the parathyroid glands in healthy young subjects, while the oxyphile cells appear with increasing age and increases further dramatically in number in patients with CKD. This oxyphile cell type is characterized by high expression of PTH and PTHrP, making future studies on the role of PTHrP in uremic parathyroids very relevant.
Conclusion Plasma PTHrP concentrations were clearly measurable in uremic patients on chronic hemodialysis and corresponded positively to age. Plasma PTHrP did not correlate to plasma PTH concentration neither in all patients on hemodialysis nor in those with hypocalcemia and as such stimulated PTH secretion. This might indicate that the enhanced expression in uremia of PTHrP in the parathyroid glands in secondary hyperparathyroidism, might not directly be transferred to the circulation, but would theoretically be of importance for the autocrine/paracrine functions within the enlarged parathyroid glands.
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Acknowledgements The authors thank technician Kirsten Bang for her excellent contribution.
Funding sources No external funding sources were received. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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