INTRODUCTION

High phosphate is a key factor in the pathogenesis of secondary hyperparathyroidism. In addition, hyperphosphatemia has been shown to be a significant predictor of mortality among dialysis patients (1,2).

Phosphate and PTH secretion and synthesis

Recent in vivo and in vitro studies show that high phosphate directly stimulates parathyroid hormone (PTH) secretion (3-10). Almaden et al (3) demonstrated that intact rats parathyroid glands incubated in a high phosphate medium increased PTH secretion despite no change in the ionized calcium concentration in the medium. This study was the first report of a direct in vitro effect of phosphate on PTH secretion. Previous attempts to demonstrate an in vitro effect of phosphate on PTH secretion using dispersed parathyroid cells failed to show an effect of phosphate on PTH secretion (11).

In fact Nielsen et al (12) showed that intanct parathyroid tissue rather than dispersed cells was required to see the in vitro efect of phosphate on PTH secretion and Slatopolsky et al (5) proved that when intact parathyroid glands were used there is an effect of phosphate on PTH secretion in vitro. In vivo studies by Kilav R et al (4) and Hernandez et al (7) showed in rats that dietary induced high serum phosphate produced an elevation in serum PTH and an increase in PTH mRNA. This effect could not be explained by changes in calcium or calcitriol.

High serum phosphate levels are observed in patients with end stage renal disease; we were able to show the stimulation of PTH secretion and PTHmRNA by high phosphate in vitro in hyperplastic parathyroid tissue obtained at the time of parathyroidectomy from hemodilaysis patients with severe hyperparathyroidism (8). Further demonstration of a direct effect of phosphate on PTH secretion was obtained in two in vivo studies in hemodialysis patients (9) and in dogs (10) respectively.

The mechanism by which the parathyroid cell senses changes in serum phospahte is unknown. A parathyroid cell membrane phosphate cotransporter was cloned (13); the synthesis of this protein is modulated by changes in the dietary content of phosphate, and it has been proposed that this transpoter may function as a putative "phosphate sensor" for the parathyroid cell (14) . However, little is known about the intracellular events that mediate the regulation of PTH secretion by phosphate.

By contrast, the early signal transduction mechanisms involved in the stimulation of PTH release by low extracellular calcium are increasingly understood. In order to understand intracellular mechanisms by which phosphate may affect PTH secretion it is appropriate to summarize the intracellular signals that mediate the chamge in PTH secretion in response to extracellular calcium. Extracellular calcium concentration modulates PTH secretion via a G-protein-coupled calcium-sensing receptor (15). This effector system includes the hydrolysis of membrane phospholipids by phospholipase C (PLC), phospholipase D (PLD), and phospholipase A2 (PLA2) to generate the appropiate intracellular signals (16).

High extracellular calcium is coupled to the activation of PLA2 and the formation of arachidonic acid (AA), a potent inhibitor of PTH release, which acts via the 12- and 15-lipoxygenase pathway (17,18). The precise mechanisms by which high extracellular calcium stimulates PLA2 activity in parathyroid cells are not totally clear although a recent work suggests that mitogen-activated protein kinases (MAP Kinases) are involved in PLA2 activation (19). In other cells, the level of intracellular calcium increases in response to calcium receptor dependent PLC activation; however, it is not known whether an elevation of intracellular calcium stimulates PLA2 activity, which could be a reasonable mechanism whereby PLA2 activity is coupled to the activation of PLC.

The results of an in vitro study in parathyroid tissue (3) showed that despite the presence of high phosphate concentration in the medium, the addition of exogenous AA to the medium restored the capacity of a high calcium concentration the inhibit PTH secretion. Therefore, the addition of AA reversed the stimulatory effect of phosphate on PTH secretion. In a different study, Almaden et al (20) demonstrated that the increasen in PTH secretion induced by high extracellular phosphate was associated to a decrease in AA production by parathyrioid cell. Thus high phosphate may stimulates PTH secretion by inhibiting AA production.

The inhibition of AA production by phosphate was tissue specific since in glomerulosa cells, which increase AA production in response to angiotensin II, the addition of phosphate to the medium did not affect AA production. In a more recent work (21) we have shown that in rat parathyroid tissue an increase in intracellular calcium (by enhancing calcium entry to the cell or by stimulating the calcium release from intracellular stores), activates PLA2 resulting in increased AA production; this results explain why in parathyroid cells the increase in intracellular calcium (which normally occurs in response to high extracellular) produces an inhibition of PTH secretion.

Since previously we have shown that AA production by parathyroid cells was decreased by a high extracellular phosphate (20), we evaluated the effect of an elevation of intracellular calcium on AA production in the presence of high extracellular phosphate. The results demonstrated that despite high phosphate in the medium the elevation of intracellular calcium was capable of inducing a marked increase in AA production which resulted in a decrease in PTH secretion.

The elevation of cytosolic calcium was able to prevent the stimulation of PTH secretion by phosphate. These results support the hypothesis that the reduction in AA production induced by high extracellular phosphate is due to an inadequate increase in cytosolic calcium in response to stimulation of CaR by calcium.

The recent work by the group of Silver on the regulation of PTHmRNA by phosphate reveals that low phosphate, and also high calcium, increases PTHmRNA by a post-transcriptional mechanism. The PTHmRNA molecule is stabilized by the AUF-1 protein, which bind to the UTR; parathyroid cell AUF-1 is increased by low phosphate and high calcium (22 ).

Phosphate and parathyroid cell proliferation

The knowledge of the mechanisms underlaying the regulation of parathyroid cell proliferation are poorly understood. The effect of phosphate on parathyroid cell proliferation has been addressed by Nave-Many et al., (23) who showed that in uremic rats fed a low phosphate diet (LPD, P=0.02%), the parathyroid cell cycle was markedly inhibited; however in these rats, the LPD produced hypercalcemia which may decrease the proliferation independently.

In addition, Yi et al. (24) found that in uremic rats a change in the dietary content of phosphate from 0.6 to 0.3% prevented the parathyroid proliferationwithout a detectable change in serum calcium, phosphate or calcitriol. Parfitt et al. showed an increase in cell birth rate in rats after an extremely high phosphate diet (HPD, P=3.4%); in that study (25), serum calcium decreased and calcitriol increased, changes that potentially affect parathyroid cell proliferation.

A study of our laboratory (26) showed that in normal rats a HPD (P=1.2%) stimulated the parathyroid cells to progress to the S phase of the cell cylce in association with an increase in serum PTH but without changes in calcium, phosphate or calcitriol. The stimulation of the proliferation was observed as early as 24 hours after initiation of the HPD and by day 15 the cell proliferation becamed normal; these results suggest that parathyroid cell cycle reached a new steady stateonce the parathyroid gland was enlarged.

The fact that serum phosphate did not increase does not exclude the possibility of an increase in the body burden of phosphate resulting from the HPD; the phosphate accumulation may be responsible for the increase in parathyroid cell proliferation. Slatopolsky´s group has shown that in uremic rats, a HPD (P=0.8%), produced a significant enlargement of parathyroid glands even by 2 days after the induction of uremia (27,28). The same group of authors have studied the effect of phosphate on the parathyroid gland calcium receptor (CaR) (29). They found that high dietary phosphate decreases parathyroid CaR and that phosphate restriction prevented both, the development of the hyperplasia and the decrease in parathyroid CaR. Thus, they conclude that the improvement of the parathyroid function with dietary phosphate restriction in renal failure may be due, in part, to increased CaR expression.

Calcitriol is an important regulator of the parathyroid cell proliferation; in renal failure a deficiency of CTR contributes to the development of parathyroid hyperplasia (30,31). CTR inhibit parathyroid cell proliferation (32) by decreasing the expresssion of the proto-oncogene c-myc (33); however, hyperplastic tissues from severe 2ºHPT fail to respond to CTR (34,35). In a recent study in vitro, we have shown that in contrast to the normal dog, the response to CTR in human parathyroid glands from patients with 2ºHPT was marginal (36). Such a failure may be due to several factors, including a decrease in the number of the vitamin D receptors (VDR) (37-40).

The presence of hyperphosphatemia has also been accepted as other important factor in the resistance of the hyperplastic parathyroid glands to CTR (41). In a recent study (unpublished data ) we analyzed whether the rate of cell proliferation in hiperplastic parathyroid tissue from parathyroidectomized patients with 2ºHPT could be explained by any parameter of the glands or the patient at the time of the surgery. The results showed that the % cells in the S phase (a measurement of the proliferative rate) was inversely correlated with the age and female gender.

However, when the proliferative rate and the response to CTR (the inhibition of cell proliferation by CTR) of the same tissues was assesed in vitro, they showed to be inversely correlated not only with female gender, but also with the level of the pre-parathyroidectomy serum phosphate. Previous reports have also pointed out the neccesity of a control of serum phosphate to achieve a a proper response of the PTH secretion to CTR in uremic patients (42).

In a recent study in azotemic rats, Dusso et al have shown that a high phosphate diet increases parathyroid cell proliferation by increasing TGF-a whereas a low phosphate diet decreased parathyroid cell proliferation by stimulating the expression of p21/WAF; however they could not stablish a relationship between the high phosphate and the expression of p21 (43). CTR is known to decrease cell proliferation by inhibitig c-myc, which then result in the stimulation of p21. Thus, it is possible that the dietary phosphate loading could overcome the stimulatory effect of CTR on p21 to stimulate the parathyroid cell proliferation.

REFERENCES

1- Block GA, Hulbert-Shearon TE, Levin NW et al. Association of serum phosphate and calcium x phosphorus product with mortality risk in chronic hemodialysis patients: A national study. Am J Kidney Dis 31: 607-617, 1998

2- Goodman WG, Goldin J, Kuizon BD et al. Coronary artery calcification in young adults with end-stage renal disease undergoing dialysis. N Engl J Med 342: 1478-1483, 2000

3- Almaden Y, Canalejo A, Hernandez A, Ballesteros E, Garcia-Navarro S, Torres A, Rodriguez M. Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 11:970-976, 1996.

4- Kilav R, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 96: 327-333, 1995.

5- Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, MacDonald P, Brown A. Phosphate (PO4) restriction prevents parathyroid cell growth in uremic rats and high phosphate directly stimulates PTH secretion in tissue culture. J Clin Invest 97: 2534-2540, 1996.

6- Nielsen PK, Feldt-Rasmusen U, Olgaard K. A direct effect of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol Dial Transplant 11:1762-1768, 1996.

7- Hernandez A, Concepcion MT, Rodriguez M, Salido E, Torres A. High phosphate diet increases prepro PTH mRNA independent of calcium and calcitriol in normal rats. Kidney Int 50: 1872-1878, 1996.

8- Almaden Y, Hernandez A, Torregrosa V, Canalejo A, Sabate L, Fernandez Cruz L, Campistol JM, Torres A, Rodriguez M. High phosphate directly stimulates PTH secretion and synthesis by human parathyroid tissue. J Am Soc Nephrol 9:1845-1852, 1998.

9- de Francisco ALM, Cobo MA, Setien MA, Rodrigo E, Frsenedo GF, Unzueta MT, Amado JA, Ruiz JC, Arias M, Rodriguez M. Effect of serum phosphate on parathyroid hormone secretion during hemodialysis. Kidney Int 54: 2140-2145, 1995.

10- Estepa JC, Aguilera-Tejero E, Lopez I, Almaden Y, Rodriguez M, Felsenfeld AJ. Effect of phosphate on PTH secretion in vivo. J Bone Min Res 14:1848-1854, 1999

11- Slatopolsky E, Finch J, Ritter C, Zhong M, Denda M, Dusso A, MacDonald P Brown A: Dietary phosphorus restriction suppresses pre-pro PTH mRNA independent of 1,25-(OH)2 D3 and ionized calcium in renal failure [Abstract]. J Am Soc Nephrol 1994, 5:889.

12- Nielsen PK, Feldt-Rasmussen U, Olgaard K: A direct effect in vitro of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersesd parathyroid cells. Nephrol Dial Transplant 11:1762-1768, 1996.

13- Tatsumi S, Segawa H, Morita K, Haga H, Kouda T, Yamamoto H, Inoue Y, Nii T, Katai K, Taketani Y, Miyamoto K, Takeda E. Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands. Endocrinology 139: 1692-1699, 1998.

14- Miyamoto K, Tatsumi S, Morita K, Takeda E. Does the parathyroid "see" phosphate? Nephrol Dial Transplant 13: 2727-2729, 1998.

15- Brown EM, Gamba G, Riccardi D, Lombardi D, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca 2+-sensing receptor from bovine parathyroid. Nature 366: 575-580, 1993.

16- Kifor O, Díaz R, Butters R, Brown EM: The Ca2+-sensing receptor (CaR) activates phospholipases C, A2 and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J Bone Miner Res 12: 715-725, 1997.

17- Bourdeau A, Souberbielle J-C, Bonnet P, Herviaux P, Sachs C, Lieberherr M. Phospholipase-A2 action and arachidonic acid metabolism in calcium-mediated parathyroid hormone secretion. Endocrinology 130: 1339-1344, 1992.

18- Bourdeau A, Moutahir M, Souberbielle JC, Bonnet P, Herviaux P, Sachs C, Lieberherr M. Effects of lipoxygenase products of arachidonic metabolism on parathyroid hormone secretion. Endocrinology 135: 1109-1112, 1994.

19- Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchy T, Yao T, Kifor I, Brown EM. Regulation of MAPKinase by calcium-sensing receptor in bovine parathyroid and CaR transfected HEK293 cells. Am J Physiol Renal Physiol 280: F291-F302, 2001.

20- Almaden Y, Canalejo A., Ballesteros E., Añón G., Rodriguez M. Effect of high extracellular phosphate concentration on arachidonic acid production by parathyroid tissue in vitro. J Am Soc Nephrol 11: 1712-1718, 2000.

21- Almaden Y., Canalejo A., Ballesteros E., Añon G., Cañadillas S., Rodriguez M. The regulation of arachidonic acid production by intracellular calcium, in parathyroid cells: the effect of extracellular phosphate. J Am Soc Nephrol, 2001 (In press).

22- Sela-Brown A., Silver J., Brewer G., Naveh-Many T. Identification of AUF1 as a parathyroid hormone mRNA 3’-untranslated region-binding protein that determines parathyroid hormone mRNA stability. J Biol Chem 275 (10): 7424-7429, 2000.

23- Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats. The effect of calcium, phosphate and vitamin D. J Clin Invest 1995, 96:1786-1793

24-Yi H, Fukugawa M, Yamato H, Kumagai M, Watanabe T, Kurokawa K: Prevention of enhanced parathyroid hormone secretion synthesis and hyperplasia by mild dietary phosphorus restriction in early chronic renal failure rats: Possible direct role of phosphorus. Nephron 1195, 70:242-248

25- Wang Q, Palnitkar S, Parfitt AM. Parathyroid cell proliferation in the rat: effect of age and phosphate administration and recovery. Endocrinology 137: 4558-4562, 1996

26- Canalejo A, Hernandez A, Almaden Y, Concepcion MT, Felsenfeld A, Torres A, Rodriguez M: The effect of a high phosphorus diet on the parathyroid cell cycle. Nephrol Dial Transplant 13:19-22, 1998

27- Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, MacDonald P, Brown A: Phosphorus restriction prevents parathyroid gland growth. High phosphate directly stimulates PTH secretion in vitro. J Clin Invest 97:2534-2540, 1996

28- Denda M, Finch J, Slatopolsky E. Parathyroid hyperplasia and secondary hyperparathyroidism develop rapidly in experimental uremic rats. J Bone Miner Res 10: 5276, 1995

29- Ritter C, Finch J, Slatopolsky E, Brown AJ. The decrease in the calcium receptor in parathyroid glands of uremic rats is an early event in the development of secondary hyperparathyroidism. J Am Soc Nephrol (10): 623A, 1999 (Abstract)

30- Llach F, Massry SG: On the pathogenesis of secondary hyperparathyroidism in moderate renal insufficiency. J Clin Endocrinol Metab 61:601-606, 1985

31- Feinfeld DA, Sherwood LM: Parathyroid hormone and calcitriol in chronic renal failure. Kidney Int 33:1049-1058, 1988

32- Szabo A, Merke J, Beier E, Mall G, Ritz E: 1,25(OH)2 Vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int 35:1049-1056, 1989

33- Kremer R, Bolivar I, Goltzman D, Hendy GN: Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 125:935-941, 1989

34- Drueke T: The pathogenesis of parathyroid gland hyperplasia in chronic renal failure. Kidney Int 48:259-272, 1995

35- Fukagawa M, Kitaoka M, Kurokawa K: Resistance of the parathyroid glands to vitaminD in renal failure: Implications for medical management. Kidney Int 52: S60-S64, 1997

36- Canalejo A, Almaden Y, Torregrosa V, Gomez-Villamandos JC, Ramos B, Campistol JM, Felsenfeld AJ, Rodriguez M: The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Am Soc Nephrol 11:1865-1872, 2000

37- Merke J, Hugel U, Zlotkowski A, Szabo A, Bommer J Mall G, Ritz E: Diminished parathyroid 1,25-(OH)2D3 receptors in experimental uremia. Kidney Int 32:350-353, 1987

38- Korkor AB: Reduced binding of [3H]1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 316:1573-1577, 1987

39- Brown AJ, Dusso A, Lopez-Hilker S, Lewis-Finch J, Grooms P, Slatopolsky E: 1,25-(OH)2D3 receptors are decreased in parathyroid glands from chronically uremic dogs. Kidney Int 35:19-23, 1989

40- Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y: Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 92:1436-1443, 1993

41- Rodriguez M, Felsenfeld AJ, Williams C, Pederson JA, Llach F: The effect of long-term intravenous calcitriol administration on parathyroid function in hemodialysis patients. J Am Soc Nephrol 2:1014-1020, 1991

42- Felsenfeld AJ, Rodríguez M. Phosphorus, calcium and secondary hyperparathyroidism: an attemt to to integrate an historical and modern perspective. J Am Soc Nephrol 10: 878-890, 1999

43- Dusso AS, Pavlopoulos T, Naumovich L, Lu Y, Finch J, Brown AJ, Morrissey J, Slatopolsky E: p21WAF and transforming growth factor-a mediate dietary phosphate regulation of parathyroid cell growth. Kidney Int 59:855-865, 2001