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Cell Biology of Parathyroid Hyperplasia in Uremia
Cell Biology of Parathyroid Hyperplasia in Uremia MASAFUMI FUKAGAWA, MO, PHD
ABSTRACT: Marked parathyroid hyperplasia of heterogeneous degrees is often seen in chronic dialysis patients with severe secondary hyperparathyroidism. In uremia, parathyroid cell proliferation is initially stimulated by decreased concentration of calcium ions and calcitriol and also by direct effect of phosphate accumulation, leading to diffuse hyperplasia ofthe parathyroid. Then, small nodules caused by monoclonal cell proliferation form within diffuse hyperplasia, which progress to form nodular hyperplasia. Cells in nodular hyperplasia have a lower density of calcitriol receptor and calcium-sensing receptor than diffuse hyperplasia and are thus more resistant to medical therapy, including calcitriol pulse therapy. One of these nodules may grow more vigorously than the others and may finally occupy a large part of the enlarged gland. Genetic mutations and rearrangements of these cells in nodular hyperplasia remain to be fully elucidated in the near future to establish an effective method for the prevention of parathyroid hyperplasia in uremia. KEY INDEXING TERMS: Parathyroid; Diffuse hyperplasia; Nodular hyperplasia; Calcitriol; Calcium; Phosphate [Am J Med Sci 1999;317(6):377-82.]
M
arked enlargement of the parathyroid glands is one of the characteristic features of chronic dialysis patients with severe secondary hyperparathyroidism. 1 ,2 Solitary adenoma is responsible for more than 80% of primary hyperparathyroidism, whereas multiple hyperplasia is the commonest abnormality in uremic patients, which suggests the different pathogeneses of parathyroid growth in these two types of hyperparathyroidism. 3 Heteroge-
From the Division of Nephrology, Tokyo Teishin Hospital and University of Tokyo School of Medicine, Tokyo, Japan. Correspondence: Masafumi Fukagawa, M.D., Ph.D., Division of Nephrology, Tokyo Teishin Hospital, 2-14-23 Fujimi, Chiyoda-ku, Tokyo 102-8798, Japan (E-mail: [email protected]). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
neous and asymmetric growth of glands in the same patient is common in uremic patients. As shown in Figure 1, the size of four (or more) parathyroid glands in the same patients varies so much that the different characters of the component cells of each gland are strongly suggested. The pathogenesis of parathyroid hyperplasia has been recently elucidated at the cellular and molecular levels 4 ,5 because of the recent development of alternative research methods, but also thanks to several careful clinical observations. In this review, I would like to summarize the cell biology of parathyroid hyperplasia and also discuss its clinical implications. Regulation of Parathyroid Cell Proliferation
Under normal conditions, the turnover rate of parathyroid cells is extremely low. 2 However, they do have the potential to replicate if provided the appropriate stimulation. As discussed in other articles in this issue, several abnormal factors can serve as stimuli for parathyroid cell proliferation in uremia. These include decreased plasma concentrations of ionized calcium and 1,25-dihydroxyvitamin D3 (calcitriol) and the accumulation ofphosphate. 6 The contribution of each factor to the development of secondary hyperparathyroidism has been evaluated experimentally both in vivo and in vitro, but there remain some points of controversy, probably because of the absence of established parathyroid cell lines and of model animals with a very long history of uremia, which simulates chronic dialysis patients. It has not been fully elucidated how the stimulation of parathyroid hormone (PTH) secretion and synthesis in uremia leads to hypertrophy and hyperplasia of parathyroid cells. Difference or transition between parathyroid cell hypertrophy and hyperplasia is another problem to be clarified. 2 In weanling rats, hypocalcemia induced by a lowcalcium diet leads to vigorous proliferation of parathyroid cells. 7 Such effects of calcium concentration on cell proliferation were also demonstrated in vitro with primary culture cells of parathyroid by one group of researchers,s but the results were not confirmed by another groUp.9 The physiological rele377
Parathyroid Hyperplasia in Uremia
in addition to its effects on PTH secretion and synthesis, as shown recently.1 3,14 Although calcium ions and calcitriol obviously play roles in several steps of the apoptotic process, the role of apoptosis in the development and regression of parathyroid hyperplasia is totally in chaos at the momentJ 5 This is partly because of the sensitivity of the methods used to demonstrate apoptosis directly or indirectly and also because of the difference between animal models and humans. Most studies have been unsuccessful in demonstrating apoptosis in animal models of parathyroid hyperplasia, even with treatment or diet modifications. 7 ,16 In contrast, several groups did demonstrate apoptosis in normal and abnormal human parathyroid,17,18 They also examined the expression of Bcl-2 and/or Bax, key proteins that prevent or promote apoptosis. Uda et al demonstrated that, with increased expression ofBcl-2, the number of apoptotic cells was lower in secondary hyperparathyroidism than in primary hyperparathyroidism. 18 They suggested that the remarkable proliferation of parathyroid cells in secondary hyperparathyroidism may be related to the reduction of apoptosis of parathyroid cells by Bcl-2 expression, whereas another group reported an increase of apoptosis, which may counteract the elevated proliferation rates of parathyroid cells. 19 Despite such controversies, clinical observations and recent data using human samples strongly support the role of these three factors for the development of parathyroid hyperplasia in uremia as will be discussed in the next two sections. Figure 1. Enlarged parathyroid glands in uremia. Note the marked difference of size among three glands surgically excised from one patient. The smallest gland was used for autotransplantation and not included in this figure.
vance of these in vitro data remains a matter of dispute, because the number of calcium-sensing receptors on these cells has been shown to decrease rapidly. 10 Suppression of parathyroid cell proliferation by calcitriol has been demonstrated by several groups with the use of primary culture cells. 9 Although [3Hlthymidine incorporation into parathyroid cells excised from uremic rats was decreased by calcitriol administration as shown by Szabo et al,l1 NavehMany et al failed to demonstrate a decrease of proliferative cell nuclear antigen (PCNA)-positive cells by calcitriol injection in vivo. 7 The effects of phosphate on parathyroid cell proliferation has been recently examined intensively. Parathyroid cell growth was stimulated by phosphate load and was prevented by phosphate restriction in uremic rats without any changes in calcium and calcitriol concentration. 12 ,13 Thus, phosphate may regulate parathyroid cell proliferation directly
378
Development and Progression of ParathyrOid HyperplaSia in Uremia
It has long been recognized anecdotally that the size of parathyroid glands correlates with the duration and severity of uremia or roughly correlates with basal PTH level.2° In addition to such observations, McCarron first reported that parathyroid size correlates better with the stimulated peak PTH secretion than with the basal PTH level.2 1 This abnormality was mainly attributed to the increase of parathyroid cell number. On the other hand, Brown et al demonstrated that the characters of the cells from each gland were different (ie, the different response to the changes of calcium ion concentration).22 Thus, the number and character of the cells determine the nature of enlarged parathyroid glands in uremia. Parathyroid hyperplasia in uremic patients is histologically divided into two types: diffuse hyperplasia with normal lobular constitution, and nodular hyperplasia with a well-circumscribed nodule surrounded by a fibrous capsule. 23 These two types of hyperplasia have several different features; nodular hyperplasia is considered the more progressed type of hyperplasia. Nodular hyperplasia is usually seen June 1999 Volume 317 Number 6
Fukagawa
in larger and heavier glands in patients with severe secondary hyperparathyroidism. Shift of set point is more severe in nodular hyperplasia than in diffuse hyperplasia. 24 Furthermore, when examined using DNA analysis, cells in nodular hyperplasia have higher proliferative potential than diffuse hyperplasia. 25 ,26 What, then, is the nature of these different cell characters in nodular and diffuse hyperplasia? The first candidate is the decrease of calcitriol receptor density initially shown by Korkor. 27 The number of calcitriol receptors was more decreased in nodular hyperplasia than in diffuse hyperplasia. 2s Such a difference was evident among glands obtained from the same patient. Small nodules forming within diffuse hyperplasia tend to have a lower density of calcitriol receptors compared with those of the surrounding tissue. Cells with more severe decreases in calcitriol receptor numbers then proliferate more vigorously to eventually form nodular hyperplasia. Furthermore, calcitriol receptor density correlated inversely with both the weight of enlarged glands and the number of PCNA-positive cells, which supports the contribution of this abnormality to parathyroid hyperplasia (Figure 2).29 Recently, a decrease of calcium-sensing receptors has been reported in the parathyroid of dialysis patients; this decrease was more severe in nodular hyperplasia than in diffuse hyperplasia. 3o ,31 Monoclonal cell proliferation is strongly suggestive of tumorous growth and has been demonstrated by Arnold et al in adenomas of primary hyperparathyroidism using X-chromosome inactivation analysis. 32 They also confirmed the frequent presence of monoclonal cell proliferation in markedly enlarged parathyroid glands in dialysis patients. 33 Tominaga et al further studied this issue by extracting DNA from each nodule and clearly demonstrating that all nodules showed monoclonal proliferation, irrespective of their size, and that the origin of each nodule is different. 34 Thus, a critical change of the characters of parathyroid cells occurs at the early stage of hyperplasia (ie, early nodularity in diffuse hyperplasia). Mutations or translocations of specific genes responsible for tumorous growth of parathyroid have been reported in primary hyperparathyroidism. 4,35 Loss or inactivation of tumor suppressor genes has been also shown to be important. PRAD1/cyclin D is a well established oncogene in which the 5 '-promoter sequence of the PTH gene is translocated immediately upstream of the cyclin D gene. 35 Thus, the stimulation of PTH gene transcription evokes the overexpression of cyclin D, leading to tumorous proliferation of parathyroid cells. In addition, mutations ofthe retinoblastoma gene, the p53 suppressor gene, the multiple endocrine neoplasia type 1 (MEN-l) gene, the RET oncogene, etc., have also been suggested in adenoma. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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Figure 2. Calcitriol receptor density and parathyroid cell proliferation in uremia, Note the significant difference of calcitriol receptor density (0) and the number of PCNA-positive cells between nodular and diffuse hyperplasia. (From Koike T, Fukuda N, Fukagawa M, et al. Correlation of enhanced cell proliferation with decreased density of vitamin D receptor in situ in parathyroid hyperplasia in chronic dialysis patients. Nephrology 1997;3: 279-84. Copyright © 1997 Blackwell Science Ltd. Modified and reproduced with permission.)
In uremic hyperparathyroidism, Falchetti et al demonstrated the allelic loss of 11q13 36 ; other groups also reported abnormalities in chromosome 11,37 on which the MEN-l gene is 10cated. 3s In addition, Nomura et al detected more frequent chromosomal aberrations (chromosomes 7 and 22) using G-banded analysis. 39 In the future, responsible genes will be identified within these loci and will help to develop new diagnostic tests. Recently, Inagaki et al analyzed genomic DNA in the parathyroid of uremic patients for possible rearrangement or allelic losses of several gene markers located on chromosome 11 p near the PTH gene and found loss of heterozygosity of the Ha-ras locus (9%) and the allelic loss of the WT -1 gene. 40 Mutation ofthe calcium-sensing receptor gene is responsible for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism with marked parathyroid hyperplasia. 41 Although the decrease in calcium-sensing receptor was confirmed in the parathyroid glands in uremic patients, Hosokawa et al could not find mutation of this gene within the coding region. 42 ,43 In summary, progression of parathyroid hyperplasia can be schematically presented in Figure 3. Within diffuse hyperplasia, cells with a lower density of calcitriol receptors (and possibly calciumsensing receptors) begin to proliferate monoclonally (early nodularity in diffuse hyperplasia) and form nodules surrounded by fibrous capsules (nodular hyperplasia). Thus, each nodular hyperplastic gland is initially composed of several monoclonal nodules of different origin. One of these nodules may grow 379
Parathyroid Hyperplasia in Uremia
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Figure 3. Progression of parathyroid hyperplasia in uremia.
much more vigorously than the others and may finally occupy a large part of the enlarged gland (single nodular gland). In a small number of patients, gene rearrangement may occur in some cells, probably within nodular hyperplasia, leading to autonomous proliferation. To understand the whole story of the progression of parathyroid hyperplasia in uremia, two questions need be answered. The first question is 'What is the abnormality responsible for monoclonal cell proliferation?' This kind of abnormality should take place frequently; thus, it may be very subtle. The second question is 'What are the biochemical and molecular mechanisms responsible for the differences of proliferative potency among these nodules?' Until these questions are answered and the underlying abnormalities are correctable, the best strategy for the management of parathyroid hyperplasia is to prevent the progression of hyperplasia at the stage of diffuse hyperplasia. Regression of parathyroid hyperplasia could be considered the reverse process of parathyroid hyperplasia. This was first recognized as a very slow process in kidney transplant recipients 44 ; however,
380
several groups have suggested that more rapid regression of parathyroid hyperplasia may be induced by calcitriol pulse therapy45-48 with some controversies. 49 In our experience, parathyroid hyperplasia regresses in all cases ifPTH secretion is successfully suppressed. 46 Despite these reports, the mechanism of such regression remains to be elucidated. In animal models of chronic renal failure, the same group of researchers reported contradictory results on the decrease of parathyroid weight by calcitriol therapy.ll.50 Contribution of apoptosis has not been fully supported in model animals. 7 In some cases, spontaneous hemorrhage within the gland may have contributed in the regression of parathyroid hyperplasia, which phenomenon has been recently recognized more often than before in both primary and secondary hyperparathyroidism. 51 Clinical Implications of the Pathogenesis of ParathyrOid Hyperplasia
It has long been suggested that chronic dialysis patients with severe hyperparathyroidism have multiple enlarged parathyroid glands. By serial and routine evaluation of parathyroid size by ultrasonography, we have revealed that the response to calcitriol pulse therapy depends on the size of parathyroid glands rather than on the degree of PTH hypersecretion. 52 The critical size in our study was 1 cm in diameter or 0.5 cm3 in volume detected by ultrasonography, which size roughly corresponds to 0.5 g in weight. Autotransplanted fragments from glands heavier than this weight have been also reported to relapse more frequently than smaller glands. 26 This 'critical size of the glands' has several cellular and molecular reasons. Histologically, 90% of glands of this size show nodular hyperplasia with lower densities of calcitriol receptors and calciumsensing receptors than diffuse hyperplasia. Thus, parathyroid cells in glands larger than 0.5 cm3 are refractory to active vitamin D therapy.53,54 Based upon these pathophysiological models, we have developed two new techniques with favorable results as adjuncts to calcitriol pulse therapy. 55 The first technique is selective percutaneous ethanol injection therapy. In this protocol, we selectively destroy all glands larger than 0.5 cm3 that should be resistant to calcitriol pulse therapy, thus leaving only small glands responsive to calcitriol,56 For the selection of the glands to be destroyed, it is also useful to evaluate blood supply to the hyperplastic glands by color Doppler flow mapping. 3 A second technique is direct calcitriol injection. 57 Theoretically, higher concentration of calcitriol should be more effective in suppressing the function of parathyroid glands with lower density of calcitriol receptors; however, higher doses of calcitriol usually lead to marked hypercalcemia. To achieve a very June 1999 Volume 317 Number 6
Fukagawa
high concentration of calcitriol only locally within parathyroid glands, we repeatedly injected calcitriol solution (1 ILg/mL) directly into enlarged glands larger than 0.5 cm3 . Direct calcitriol injections suppressed PTH hypersecretion and restored the responsiveness to calcitriol in chronic dialysis patients. Thus, very high local concentration of calcitriol not only suppressed the function of parathyroid cells with a lower density of calcitriol receptor but may also have up-regulated calcitriol receptor density in parathyroid cells. 58 Concluding Remarks
As can be seen, an increasing volume of information has been accumulating on the cellular and molecular pathogenesis of parathyroid hyperplasia in uremia. Because established nodular hyperplasia is refractory to all modes of medical therapy available, it is really important to prevent the progression of diffuse hyperplasia to nodular hyperplasia. Effective strategy on this step needs to be established in the near future. 59
13.
14. 15. 16. 17.
18. 19. 20. 21.
References 1. Coburn JW, Llach F. Renal osteodystrophy. In: Narins RG, editor. Maxwell & Kleeman's clinical disorders of fluid and electrolyte metabolism. 5th ed. New York: McGraw Hill; 1994. p. 1299-377. 2. Parfitt AM. Parathyroid growth: normal and abnormal. In: Bilezikian JP, Marcus R, Levine MA, editors. The parathyroids: basic & clinical concepts. New York: Raven Press; 1994. p.373-406. 3. Fukagawa M, Kitaoka M, Inazawa T, et al. Imaging ofthe parathyroid in chronic renal failure: diagnostic and therapeutic aspects. CUIT Opin Nephrol Hypertens 1997;6:349-55. 4. Tominaga Y, Takagi H. Molecular genetics of hyperparathyroid disease. CUIT Opin Nephrol Hypertens 1996;5:33641. 5. Driieke TB. The pathogenesis of parathyroid gland hyperplasia in chronic renal failure. Kidney Int 1995;48:259-72. 6. Akizawa T, Fukagawa M, Koshikawa S, et al. Recent progress in management of secondary hyperparathyroidism. Curr Opin Nephrol Hypertens 1993;2:558-65. 7. Naveh-Many T, Rachmaninov R, Livni N, et al. Parathyroid cell proliferation in normal and chronic renal failure rats: the effects of calcium, phosphate and vitamin D. J Clin Invest 1995;96:1786-93. 8. Bianchi S, Fabiani S, Muratori M, et al. Calcium modulates the cyclin D1 expression in a rat parathyroid cell line. Biochem Biophys Res Commun 1994;204:691-700. 9. Kremer R, Bolivar I, Goltzman D, et al. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinol 1989;125:935-41. 10. Brown AJ, Zhong M, Ritter C, et al. Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression. Biochem Biophys Res Commun 1995;212:861-7. 11. Szabo A, Merke J, Beier E, et al. E3 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int 1989;35:1049-56. 12. Yi H, Fukagawa M, Yamato H, et al. Prevention of enhanced parathyroid hormone secretion, synthesis and hyperplasia by mild dietary phosphorus restriction in early chronic THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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abnormal parathyroid hormone genes in parathyroid adenoma. N Engl J Med 1988;318:658-62. Arnold A, Brown MF, Urena P, et al. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia, J Clin Invest 1995;95:2047-53. Tominaga Y, Kohara S, Namii Y, et al. Clonal analysis of nodular hyperplasia in renal hyperparathyroidism, World J Surg 1996;20:744-52. Arnold A. Genetic basis of endocrine disease 5: molecular genetics of parathyroid gland neoplasia. J Clin Endocrinol Metab 1993;77:1108-12. Falchetti A, Bale AE, Amorosi A, et aI. Progression of uremic hyperparathyroidism involves allelic loss on chromosome 11. J Clin Endocrinol Metab 1993;76:139-44. Farnebo F, Teh BT, Dotzenrach C, et aI. Differential loss of heterozygosity in familial, sporadic, and uremic hyperparathyroidism. Hum Genet 1997;99:342-9. Heppner C, Kester MB, Agarwal SK, et al. Somatic mutation of the MENI gene in parathyroid tumors. Nat Genet 1997;16:375-8. Nomura S, Sonoo H, Tokura T, et al. Cytogenesis of secondary hyperparathyroidism [abstract]. J Am Soc N ephrol 1997;8:578A. Inagaki C, Dousseau M, Pacher N, et al. Structural analysis of gene marker loci on chromosome 10 and 11 in primary and secondary uremic hyperparathyroidism. Nephrol Dial Transplant 1998;13:350-7. Pollak M, Brown EM, Chou Y-HW, et al. Mutation in the Ca 2+ -sensing receptor gene causes familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297-303. Hosokawa Y, Pollak MR, Brown EM, et al. Mutational analysis of the extracellular Ca 2+ -sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995; 80:3107-10. Thompson DB, Samowitz WS, Oldelberg S, et al. Genetic abnormalities in sporadic parathyroid adenomas: loss ofheterozygosity for chromosome 3q markers flanking the calcium receptor locus. J Clin Endocrinol Metab 1995;80:3377-80. Parfitt AM. Hypercalcemic hyperparathyroidism following renal transplantation: differential diagnosis, management, and implications for cell proliferation control in parathyroid glands. Mineral Electrolyte Metab 1982;8:92-119. Fukagawa M, Okazaki R, Takano K, et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med 1990;323:421-2. Fukagawa M, Fukuda N, Yi H, et al. Derangement of
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parathyroid function in renal failure: Biological and clinical aspects. J Nephrol 1996;9:219-24. Hyodo T, Koumi T, Ueda M, et al. Can orall,25(OH)2Da therapy reduce parathyroid hyperplasia? Nephron 1991;59: 171-2. Cannella G, Bonucci E, Rolla D, et aI. Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. Kidney Int 1994;46:1124-32. Quarles LD, Yohay DA, Carroll BA, et aI. Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 1994;45:1710-22. Reichel H, Szabo A, Ubi J, et al. Intermittent versus continuous administration of 1,25-dihydroxyvitamin Da in experimental renal hyperparathyroidism. Kidney Int 1993; 44:1259-65. Nylen E, Shah A, Hall J. Spontaneous remission of primary hyperparathyroidism from parathyroid apoplexy. J Clin Endocrinol Metab 1996;81:1326-8. Fukagawa M, Kitaoka M, Yi H, et al. Serial evaluation of parathyroid size by ultrasonography is another useful marker for the long-term prognosis of calcitriol pulse therapy in chronic dialysis patients. Nephron 1994;68:221-8. Tominaga Y, Sato K, Tanaka Y, et aI. Histopathology and pathophysiology of secondary hyperparathyroidism due to chronic renal failure. Clin NephrolI995;44:S42-7. Tominaga Y, Tanaka Y, Sato K, et al. Histology, pathophysiology, and indications for surgical treatment of renal hyperparathyroidism. Semin Surg OncolI997;13:78-86. Kitaoka M, Fukagawa M, Ogata E, et al. Reduction of functioning parathyroid cell mass by ethanol injection in chronic dialysis patients. Kidney Int 1994;46:1110-17. Fukagawa M, Kitaoka M, Kurokawa K. Resistance to vitamin D in chronic renal failure. Kidney Int 1997;52(Suppl 62):S60-4. Kitaoka M, Fukagawa M, Kurokawa K. Direct injection of calcitriol into parathyroid hyperplasia in chronic dialysis patients with severe parathyroid hyperfunction. Nephrology 1995;1:563-8. Naveh-Many T, Marx R, Keshet E, et al. Regulation of 1,25-Dihydroxyvitamin Da receptor gene expression by 1,25dihydroxyvitamin Da in the parathyroid in vivo. J Clin Invest 1990;86:1968- 75. Fukagawa M, Kurokawa K. Renal failure and hyperparathyroidism. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. San Diego: Academic Press; 1997. p. 1227-39.
June 1999 Volume 317 Number 6
ABSTRACT: Marked parathyroid hyperplasia of heterogeneous degrees is often seen in chronic dialysis patients with severe secondary hyperparathyroidism. In uremia, parathyroid cell proliferation is initially stimulated by decreased concentration of calcium ions and calcitriol and also by direct effect of phosphate accumulation, leading to diffuse hyperplasia ofthe parathyroid. Then, small nodules caused by monoclonal cell proliferation form within diffuse hyperplasia, which progress to form nodular hyperplasia. Cells in nodular hyperplasia have a lower density of calcitriol receptor and calcium-sensing receptor than diffuse hyperplasia and are thus more resistant to medical therapy, including calcitriol pulse therapy. One of these nodules may grow more vigorously than the others and may finally occupy a large part of the enlarged gland. Genetic mutations and rearrangements of these cells in nodular hyperplasia remain to be fully elucidated in the near future to establish an effective method for the prevention of parathyroid hyperplasia in uremia. KEY INDEXING TERMS: Parathyroid; Diffuse hyperplasia; Nodular hyperplasia; Calcitriol; Calcium; Phosphate [Am J Med Sci 1999;317(6):377-82.]
M
arked enlargement of the parathyroid glands is one of the characteristic features of chronic dialysis patients with severe secondary hyperparathyroidism. 1 ,2 Solitary adenoma is responsible for more than 80% of primary hyperparathyroidism, whereas multiple hyperplasia is the commonest abnormality in uremic patients, which suggests the different pathogeneses of parathyroid growth in these two types of hyperparathyroidism. 3 Heteroge-
From the Division of Nephrology, Tokyo Teishin Hospital and University of Tokyo School of Medicine, Tokyo, Japan. Correspondence: Masafumi Fukagawa, M.D., Ph.D., Division of Nephrology, Tokyo Teishin Hospital, 2-14-23 Fujimi, Chiyoda-ku, Tokyo 102-8798, Japan (E-mail: [email protected]). THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
neous and asymmetric growth of glands in the same patient is common in uremic patients. As shown in Figure 1, the size of four (or more) parathyroid glands in the same patients varies so much that the different characters of the component cells of each gland are strongly suggested. The pathogenesis of parathyroid hyperplasia has been recently elucidated at the cellular and molecular levels 4 ,5 because of the recent development of alternative research methods, but also thanks to several careful clinical observations. In this review, I would like to summarize the cell biology of parathyroid hyperplasia and also discuss its clinical implications. Regulation of Parathyroid Cell Proliferation
Under normal conditions, the turnover rate of parathyroid cells is extremely low. 2 However, they do have the potential to replicate if provided the appropriate stimulation. As discussed in other articles in this issue, several abnormal factors can serve as stimuli for parathyroid cell proliferation in uremia. These include decreased plasma concentrations of ionized calcium and 1,25-dihydroxyvitamin D3 (calcitriol) and the accumulation ofphosphate. 6 The contribution of each factor to the development of secondary hyperparathyroidism has been evaluated experimentally both in vivo and in vitro, but there remain some points of controversy, probably because of the absence of established parathyroid cell lines and of model animals with a very long history of uremia, which simulates chronic dialysis patients. It has not been fully elucidated how the stimulation of parathyroid hormone (PTH) secretion and synthesis in uremia leads to hypertrophy and hyperplasia of parathyroid cells. Difference or transition between parathyroid cell hypertrophy and hyperplasia is another problem to be clarified. 2 In weanling rats, hypocalcemia induced by a lowcalcium diet leads to vigorous proliferation of parathyroid cells. 7 Such effects of calcium concentration on cell proliferation were also demonstrated in vitro with primary culture cells of parathyroid by one group of researchers,s but the results were not confirmed by another groUp.9 The physiological rele377
Parathyroid Hyperplasia in Uremia
in addition to its effects on PTH secretion and synthesis, as shown recently.1 3,14 Although calcium ions and calcitriol obviously play roles in several steps of the apoptotic process, the role of apoptosis in the development and regression of parathyroid hyperplasia is totally in chaos at the momentJ 5 This is partly because of the sensitivity of the methods used to demonstrate apoptosis directly or indirectly and also because of the difference between animal models and humans. Most studies have been unsuccessful in demonstrating apoptosis in animal models of parathyroid hyperplasia, even with treatment or diet modifications. 7 ,16 In contrast, several groups did demonstrate apoptosis in normal and abnormal human parathyroid,17,18 They also examined the expression of Bcl-2 and/or Bax, key proteins that prevent or promote apoptosis. Uda et al demonstrated that, with increased expression ofBcl-2, the number of apoptotic cells was lower in secondary hyperparathyroidism than in primary hyperparathyroidism. 18 They suggested that the remarkable proliferation of parathyroid cells in secondary hyperparathyroidism may be related to the reduction of apoptosis of parathyroid cells by Bcl-2 expression, whereas another group reported an increase of apoptosis, which may counteract the elevated proliferation rates of parathyroid cells. 19 Despite such controversies, clinical observations and recent data using human samples strongly support the role of these three factors for the development of parathyroid hyperplasia in uremia as will be discussed in the next two sections. Figure 1. Enlarged parathyroid glands in uremia. Note the marked difference of size among three glands surgically excised from one patient. The smallest gland was used for autotransplantation and not included in this figure.
vance of these in vitro data remains a matter of dispute, because the number of calcium-sensing receptors on these cells has been shown to decrease rapidly. 10 Suppression of parathyroid cell proliferation by calcitriol has been demonstrated by several groups with the use of primary culture cells. 9 Although [3Hlthymidine incorporation into parathyroid cells excised from uremic rats was decreased by calcitriol administration as shown by Szabo et al,l1 NavehMany et al failed to demonstrate a decrease of proliferative cell nuclear antigen (PCNA)-positive cells by calcitriol injection in vivo. 7 The effects of phosphate on parathyroid cell proliferation has been recently examined intensively. Parathyroid cell growth was stimulated by phosphate load and was prevented by phosphate restriction in uremic rats without any changes in calcium and calcitriol concentration. 12 ,13 Thus, phosphate may regulate parathyroid cell proliferation directly
378
Development and Progression of ParathyrOid HyperplaSia in Uremia
It has long been recognized anecdotally that the size of parathyroid glands correlates with the duration and severity of uremia or roughly correlates with basal PTH level.2° In addition to such observations, McCarron first reported that parathyroid size correlates better with the stimulated peak PTH secretion than with the basal PTH level.2 1 This abnormality was mainly attributed to the increase of parathyroid cell number. On the other hand, Brown et al demonstrated that the characters of the cells from each gland were different (ie, the different response to the changes of calcium ion concentration).22 Thus, the number and character of the cells determine the nature of enlarged parathyroid glands in uremia. Parathyroid hyperplasia in uremic patients is histologically divided into two types: diffuse hyperplasia with normal lobular constitution, and nodular hyperplasia with a well-circumscribed nodule surrounded by a fibrous capsule. 23 These two types of hyperplasia have several different features; nodular hyperplasia is considered the more progressed type of hyperplasia. Nodular hyperplasia is usually seen June 1999 Volume 317 Number 6
Fukagawa
in larger and heavier glands in patients with severe secondary hyperparathyroidism. Shift of set point is more severe in nodular hyperplasia than in diffuse hyperplasia. 24 Furthermore, when examined using DNA analysis, cells in nodular hyperplasia have higher proliferative potential than diffuse hyperplasia. 25 ,26 What, then, is the nature of these different cell characters in nodular and diffuse hyperplasia? The first candidate is the decrease of calcitriol receptor density initially shown by Korkor. 27 The number of calcitriol receptors was more decreased in nodular hyperplasia than in diffuse hyperplasia. 2s Such a difference was evident among glands obtained from the same patient. Small nodules forming within diffuse hyperplasia tend to have a lower density of calcitriol receptors compared with those of the surrounding tissue. Cells with more severe decreases in calcitriol receptor numbers then proliferate more vigorously to eventually form nodular hyperplasia. Furthermore, calcitriol receptor density correlated inversely with both the weight of enlarged glands and the number of PCNA-positive cells, which supports the contribution of this abnormality to parathyroid hyperplasia (Figure 2).29 Recently, a decrease of calcium-sensing receptors has been reported in the parathyroid of dialysis patients; this decrease was more severe in nodular hyperplasia than in diffuse hyperplasia. 3o ,31 Monoclonal cell proliferation is strongly suggestive of tumorous growth and has been demonstrated by Arnold et al in adenomas of primary hyperparathyroidism using X-chromosome inactivation analysis. 32 They also confirmed the frequent presence of monoclonal cell proliferation in markedly enlarged parathyroid glands in dialysis patients. 33 Tominaga et al further studied this issue by extracting DNA from each nodule and clearly demonstrating that all nodules showed monoclonal proliferation, irrespective of their size, and that the origin of each nodule is different. 34 Thus, a critical change of the characters of parathyroid cells occurs at the early stage of hyperplasia (ie, early nodularity in diffuse hyperplasia). Mutations or translocations of specific genes responsible for tumorous growth of parathyroid have been reported in primary hyperparathyroidism. 4,35 Loss or inactivation of tumor suppressor genes has been also shown to be important. PRAD1/cyclin D is a well established oncogene in which the 5 '-promoter sequence of the PTH gene is translocated immediately upstream of the cyclin D gene. 35 Thus, the stimulation of PTH gene transcription evokes the overexpression of cyclin D, leading to tumorous proliferation of parathyroid cells. In addition, mutations ofthe retinoblastoma gene, the p53 suppressor gene, the multiple endocrine neoplasia type 1 (MEN-l) gene, the RET oncogene, etc., have also been suggested in adenoma. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
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In uremic hyperparathyroidism, Falchetti et al demonstrated the allelic loss of 11q13 36 ; other groups also reported abnormalities in chromosome 11,37 on which the MEN-l gene is 10cated. 3s In addition, Nomura et al detected more frequent chromosomal aberrations (chromosomes 7 and 22) using G-banded analysis. 39 In the future, responsible genes will be identified within these loci and will help to develop new diagnostic tests. Recently, Inagaki et al analyzed genomic DNA in the parathyroid of uremic patients for possible rearrangement or allelic losses of several gene markers located on chromosome 11 p near the PTH gene and found loss of heterozygosity of the Ha-ras locus (9%) and the allelic loss of the WT -1 gene. 40 Mutation ofthe calcium-sensing receptor gene is responsible for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism with marked parathyroid hyperplasia. 41 Although the decrease in calcium-sensing receptor was confirmed in the parathyroid glands in uremic patients, Hosokawa et al could not find mutation of this gene within the coding region. 42 ,43 In summary, progression of parathyroid hyperplasia can be schematically presented in Figure 3. Within diffuse hyperplasia, cells with a lower density of calcitriol receptors (and possibly calciumsensing receptors) begin to proliferate monoclonally (early nodularity in diffuse hyperplasia) and form nodules surrounded by fibrous capsules (nodular hyperplasia). Thus, each nodular hyperplastic gland is initially composed of several monoclonal nodules of different origin. One of these nodules may grow 379
Parathyroid Hyperplasia in Uremia
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Figure 3. Progression of parathyroid hyperplasia in uremia.
much more vigorously than the others and may finally occupy a large part of the enlarged gland (single nodular gland). In a small number of patients, gene rearrangement may occur in some cells, probably within nodular hyperplasia, leading to autonomous proliferation. To understand the whole story of the progression of parathyroid hyperplasia in uremia, two questions need be answered. The first question is 'What is the abnormality responsible for monoclonal cell proliferation?' This kind of abnormality should take place frequently; thus, it may be very subtle. The second question is 'What are the biochemical and molecular mechanisms responsible for the differences of proliferative potency among these nodules?' Until these questions are answered and the underlying abnormalities are correctable, the best strategy for the management of parathyroid hyperplasia is to prevent the progression of hyperplasia at the stage of diffuse hyperplasia. Regression of parathyroid hyperplasia could be considered the reverse process of parathyroid hyperplasia. This was first recognized as a very slow process in kidney transplant recipients 44 ; however,
380
several groups have suggested that more rapid regression of parathyroid hyperplasia may be induced by calcitriol pulse therapy45-48 with some controversies. 49 In our experience, parathyroid hyperplasia regresses in all cases ifPTH secretion is successfully suppressed. 46 Despite these reports, the mechanism of such regression remains to be elucidated. In animal models of chronic renal failure, the same group of researchers reported contradictory results on the decrease of parathyroid weight by calcitriol therapy.ll.50 Contribution of apoptosis has not been fully supported in model animals. 7 In some cases, spontaneous hemorrhage within the gland may have contributed in the regression of parathyroid hyperplasia, which phenomenon has been recently recognized more often than before in both primary and secondary hyperparathyroidism. 51 Clinical Implications of the Pathogenesis of ParathyrOid Hyperplasia
It has long been suggested that chronic dialysis patients with severe hyperparathyroidism have multiple enlarged parathyroid glands. By serial and routine evaluation of parathyroid size by ultrasonography, we have revealed that the response to calcitriol pulse therapy depends on the size of parathyroid glands rather than on the degree of PTH hypersecretion. 52 The critical size in our study was 1 cm in diameter or 0.5 cm3 in volume detected by ultrasonography, which size roughly corresponds to 0.5 g in weight. Autotransplanted fragments from glands heavier than this weight have been also reported to relapse more frequently than smaller glands. 26 This 'critical size of the glands' has several cellular and molecular reasons. Histologically, 90% of glands of this size show nodular hyperplasia with lower densities of calcitriol receptors and calciumsensing receptors than diffuse hyperplasia. Thus, parathyroid cells in glands larger than 0.5 cm3 are refractory to active vitamin D therapy.53,54 Based upon these pathophysiological models, we have developed two new techniques with favorable results as adjuncts to calcitriol pulse therapy. 55 The first technique is selective percutaneous ethanol injection therapy. In this protocol, we selectively destroy all glands larger than 0.5 cm3 that should be resistant to calcitriol pulse therapy, thus leaving only small glands responsive to calcitriol,56 For the selection of the glands to be destroyed, it is also useful to evaluate blood supply to the hyperplastic glands by color Doppler flow mapping. 3 A second technique is direct calcitriol injection. 57 Theoretically, higher concentration of calcitriol should be more effective in suppressing the function of parathyroid glands with lower density of calcitriol receptors; however, higher doses of calcitriol usually lead to marked hypercalcemia. To achieve a very June 1999 Volume 317 Number 6
Fukagawa
high concentration of calcitriol only locally within parathyroid glands, we repeatedly injected calcitriol solution (1 ILg/mL) directly into enlarged glands larger than 0.5 cm3 . Direct calcitriol injections suppressed PTH hypersecretion and restored the responsiveness to calcitriol in chronic dialysis patients. Thus, very high local concentration of calcitriol not only suppressed the function of parathyroid cells with a lower density of calcitriol receptor but may also have up-regulated calcitriol receptor density in parathyroid cells. 58 Concluding Remarks
As can be seen, an increasing volume of information has been accumulating on the cellular and molecular pathogenesis of parathyroid hyperplasia in uremia. Because established nodular hyperplasia is refractory to all modes of medical therapy available, it is really important to prevent the progression of diffuse hyperplasia to nodular hyperplasia. Effective strategy on this step needs to be established in the near future. 59
13.
14. 15. 16. 17.
18. 19. 20. 21.
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