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Stimulation of osteoclastic bone resorption in a model of glycerol-induced acute renal failure: evidence for a parathyroid hormone-independent mechanism
Bone Vol. 31, No. 4 October 2002:488 – 491
Stimulation of Osteoclastic Bone Resorption in a Model of Glycerol-Induced Acute Renal Failure: Evidence for a Parathyroid Hormone-Independent Mechanism A. GAL-MOSCOVICI, P. SCHERZER, D. RUBINGER, R. WEISS, M. DRANITZKI-ELHALEL, and M. M. POPOVTZER Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel
production rate of calcitriol,10,15,21 and a decreased calcitriol receptor concentration were recently shown in a rat model of incipient renal failure.10,22 Thus, it was shown that a reduced renal vitamin D receptor binding to the nuclear response element22 may be the underlying mechanism for vitamin D resistance in renal failure, which may lead to increased parathyroid hormone secretion. In a model of ARF due to bilateral nephrectomy Pavlovitch et al. found that serum parathyroid hormone was significantly elevated 5 h after ablation surgery.20 The bone changes found in their model of ARF included a significant increase in osteoclastic resorption. They concluded that this increase in bone resorption was partially mediated by the increased parathyroid hormone level. Whether the hypersecretion of parathyroid hormone is the only factor responsible for bone changes seen in ARF remains a matter of debate. The possibility of factors other than parathyroid hormone being involved in bone changes occuring in ARF has not been studied in detail. This study was undertaken to investigate the bone changes occurring in a model of glycerol-induced ARF and to evaluate the influence of parathyroid hormone on these changes.
This study was undertaken to evaluate the bone changes occurring in rats with acute renal failure (ARF). Acute renal failure was induced in rats 24 hours after dehydration by an intramuscular injection of glycerol. After induction of ARF, the rats were divided into two groups, one of which underwent parathyroidectomy (PTX). Rats with normal renal function, matched for age and weight, were used as controls and divided into two groups, one of them for PTX. At termination of the study blood and urine chemistry and bone histomorphometry were analyzed. Rats with glycerol-induced ARF developed bone changes compatible with mild hyperparathyroid bone disease, characterized mainly by increased osteoclastic bone resorption when compared with control rats having normal renal function. Rats with normal renal function following PTX developed bone disease showing complete suppression of forming and resorptive parameters. Rats with glycerol-induced ARF and PTX showed abolishment of all bone forming parameters, but a dramatic increase in osteoclastic resorption was apparent. Based on these observations we suggest that, in this model of glycerolinduced ARF, osteoclastic bone resorption may develop in the absence of parathyroid hormone, probably stimulated by other potent osteoclastogenic factors. (Bone 31:488 – 491; 2002) © 2002 by Elsevier Science Inc. All rights reserved.
Materials and Methods Experimental Design
Key Words: Acute renal failure (ARF); Parathyroidectomy (PTX); Rats; Osteoclastic bone resorption; Histomorphometry; Secondary hyperparathyroidism.
Male Sabra rats of the Hebrew University strain, weighing 180 –200 g, were housed in metabolic cages and allowed free access to standard pellet chow containing 0.78% calcium, 0.51% phosphorus, and tap water. Following an acclimatization period of about 1 week, the animals were divided into four groups:
Introduction Renal osteodystrophy occurs frequently in the course of chronic renal failure (CRF). Bone diseases in CRF spread over a broad spectrum of pathology ranging from hyperparathyroid to mixed and adynamic bone disease. Osteitis fibrosa cystica due to secondary hyperparathyroidism is still the most predominant form of renal osteodystrophy.6,7,12,13,26 There is evidence that secondary hyperparathyroidism also occurs in acute renal failure (ARF), even at a very early stage. An impaired calcium transport by intestine,3,11,14,30 a decreased
1. Control rats with normal renal function (sham and non-shamoperated). 2. Rats with normal renal function (sham-operated) undergoing parathyroidectomy (PTX). 3. Rats with acute renal failure (ARF). 4. Rats with ARF and PTX. ARF was induced by one intramuscular injection of 10 mg/kg body weight of 50% glycerol, following a 24 h period of dehydration. PTX was performed on the same day by hot wire cautery under light ether anesthesia. Twenty-four-hour urine collections were obtained during the last day of the experiment. Forty-eight hours after induction of
Address for correspondence and reprints: Dr. A. Gal-Moscovici, Nephrology and Hypertension Services, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. E-mail: [email protected] © 2002 by Elsevier Science Inc. All rights reserved.
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8756-3282/02/$22.00 PII S8756-3282(02)00849-9
Bone Vol. 31, No. 4 October 2002:488 – 491
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Table 1. Plasma biochemistry in control and rats with acute renal failure (ARF) and parathyroidectomy (PTX)
Control ARF PTX ARF ⫹ PTX
PCr (mol/L)
CrCl (L/min)
PCa (mEq/L)
PP (mEq/L)
PPTH (pg/mL)
46.27 ⫾ 8.4 179 ⫾ 43c 44.4 ⫾ 1.9 199 ⫾ 44d
537 ⫾ 69 125 ⫾ 39a 337 ⫾ 90 105 ⫾ 40b
4.75 ⫾ 0.09 4.58 ⫾ 0.39 2.72 ⫾ 0.24a 4.16 ⫾ 0.54d
3.5 ⫾ 0.92 3.32 ⫾ 0.38 4.53 ⫾ 0.16c 4.01 ⫾ 0.92
42.4 ⫾ 6.1 257 ⫾ 32e 13.6 ⫾ 0.67e,f 11.1 ⫾ 1.34e,f
p ⬍ 0.001 vs. control. bp ⬍ 0.05 vs. PTX. cp ⬍ 0.02 vs. control. dp ⬍ 0.01 vs. PTX. ep ⬍ 0.001 vs. control. p ⬍ 0.001 vs. ARF.
a f
ARF the animals were killed and blood samples were collected anaerobically from the abdominal aorta in 5 mL vacuum tubes for measurement of the creatinine, calcium, phosphate, and parathyroid hormone (PTH) levels. The biochemical measurements performed on an automated analyzer included determination of plasma and urine levels of creatinine, calcium, and phosphate. The PTH levels in the serum were determined by an immunoradiometric assay (IRMA) using a rat-specific PTH kit (Immutopics, Inc., San Clemente, CA). Histomorphometry Histomorphometric studies were performed on the right tibial bone. Tetracycline (Ledermycin, Lederle, Germany) at a dose of 25 mg/kg was injected intraperitoneally at 5 days and 1 day prior to killing. The duration of the study was 6 days. The tibial bones were removed, further fixed in Carson– Milloning solution,1 and embedded undecalcified in a historesin kit after dehydration in alcohol. Sections of 5 m and 10 m were cut in anter oposterior planes, using a Reichert-Jung microtome. The thicker sections (10 m) were mounted unstained for viewing of the fluorochromal marker. To identify mineralized bone, osteoid, osteoblasts, and osteoclasts, the 5 m sections were stained by the von Kossa method.5 The following parameters were calculated according to Parfitt19: 1. Osteoblast surface (Ob.S/BS), the fraction of trabecular surfaces covered by osteoblasts. 2. Osteoid surface (OS/BS), the fraction of trabecular surface covered by osteoid. 3. Osteoid volume (OV/BV), the fraction of trabecular bone volume occupied by osteoid. 4. Osteoclast surface (Oc.S/BS), the fraction of trabecular surface covered by osteoclasts. 5. Osteoclast number (Oc.N/TA), the number of osteoclasts per square millimeter of cancellous bone. 6. Osteoid thickness (O.Th), calculated as the ratio of osteoid volume to osteoid surface ⫻ 100. 7. Eroded surface (ES/BS), the fraction of trabecular surface covered by lacunae (including “active” lacunae with osteoclasts and lacunae in reversal phase; i.e., the period in which the resorption lacunae are filled with inactive-looking mononuclear cells). 8. Double-labeled tetracycline surface, the fraction of trabecular surface covered by double-labeled tetracycline. 9. Mineralized surface, the total extent of double labels plus half the extent of single labels. 10. Bone formation rate (BFR/BS) at tissue level, obtained by multiplying the mineralized surface by mineral oppositional rate. 11. Mineral appositional rate (MAR), obtained by measuring the mean distance between tetracycline labels and dividing by the number of days between doses of tetracycline.
Statistical Methods Statistical analysis of the histomorphometric data within the groups was done using nonparametric one-way analysis of variance and the SPSS statistical package. Biochemical statistical analysis was performed by the Wilcoxon rank sum test, using two-group comparison. Results are presented in the tables as mean ⫾ SE. Histomorphometry was quantified in a blinded manner by the same investigator. Random biopsy samples were assessed for verification by two different experts from Israel. The interexaminer differences were 5% for all parameters. Results Biochemical Data Rats with acute renal failure presented with increased plasma creatinine and decreased creatinine clearance when compared to rats with normal renal function (Table 1). Parathyroidectomy was documented by the presence of low calcium, high phosphate, and low PTH plasma levels. There were no changes in plasma creatinine and creatinine clearance levels in group 2 (sham-operated rats with PTX) when compared with controls (sham and non-sham). The rats with both ARF and PTX showed a decreased creatinine clearance and an increase in plasma creatinine, similar to those with ARF only. Plasma PTH level was significantly lower in the group with ARF and PTX as compared with the control group with ARF only (Table 1). Plasma calcium level in group 4 was lower than that in group 2, but the difference did not reach statistical significance. Similarly, the phosphate plasma levels in group 4 were higher than in group 2, but failed to reach statistical significance. Histomorphometric Results The histomorphometric static bone-forming parameters in the four study groups are shown in Figure 1. Rats with normal kidney function, following PTX (group 2), showed severely reduced osteoid volume and surface and absence of osteoblastic cells. These changes were statistically significant when compared with control rats or rats with ARF. In rats with ARF (group 3) a significant decrease in osteoid volume and surface, as well as in osteoblastic surface, was found when compared with control rats. The most striking suppression of osteoid volume and osteoid surface was found in the rats with both ARF and PTX (group 4). This group also showed a complete abolishment of the osteoblastic line. The dynamic bone forming parameters in groups 2, 3, and 4 showed decreased mineralizing capacity and reduced bone formation rate when compared with control rats (group 1); however, these changes were statistically nonsignificant (Figure 2). Bone-resorbing parameters of all four groups are shown in
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Figure 1. Static bone-forming parameters in control rats and in rats with acute renal failure (ARF) with and without parathyroidectomy (PTX). *P ⬍ 0.01 vs. control; **P ⬍ 0.05 vs. control; aP ⬍ 0.005 vs. PTX, ARF; b P ⬍ 0.01 vs. ARF; cP ⬍ 0.001 vs. ARF, control.
Figures 3 and 4. Rats with ARF (group 2) showed a significantly increased osteoclastic surface and osteoclast number (Figure 4) when compared with control rats (group 1). Rats with normal renal function and PTX (group 3) showed an absence of osteoclasts or osteoclastic activity. The most impressive results were observed in rats with both ARF and PTX (group 4). In this group, osteoclast number (Figure 4) and surface (Figure 3) increased 2.5-fold when compared with control rats (group 1), and was also significantly enhanced when compared to rats with ARF without PTX (group 2). Discussion The pathogenesis of renal osteodystrophy in chronic renal failure has attracted much attention. Bone disease in acute renal failure, however, has not been studied in great detail. A decrease in glomerular filtration rate (GFR) of about 40% in ARF has been reported to be associated with increased secretion of PTH.16,20,22,25 Thus, increased bone turnover with increased osteoclastic and osteoblastic activity are expected to occur. Evidence of increased osteoclastic activity, induced by bilateral nephrectomy, was reported by Pavlovitch et al. in their model of ARF, but they presented no data regarding osteoblastic activity.20 The bone abnormality in our rats with glycerol-induced ARF consisted of a significant increase in osteoclast activity, but
Figure 2. Bone formation rate in normal rats and rats with acute renal failure (ARF) with or without parathyroidectomy (PTX).
Figure 3. Osteoclastic bone resorption in normal rats and rats with acute renal failure (ARF) with or without parathyroidectomy (PTX). aP ⬍ 0.05 vs. control; bP ⬍ 0.05 vs. ARF; cP ⬍ 0.001 vs. PTX; dP ⬍ 0.001 vs. control.
differed from the classical picture of hyperparathyroid bone disease by the absence of activated osteoblasts and by the presence of depressed bone-forming parameters. Parathyroidectomy performed in these rats with ARF (group 4) showed, as expected, further reduction in osteoblastic activity. However, it was accompanied by a significant increase in osteoclastic activity when compared to rats with ARF only (group 3). This finding of increased osteoclastic number and activity in rats with ARF and PTX (group 4) suggests that factors other than PTH may be involved in osteoclast activation. The glycerol-induced model of ARF in rats is associated with an increase in endogenously produced cytokines, mainly tumor necrosis factor-␣ (TNF-␣), by activated inflammatory cells.20 –22 TNF-␣ is often implicated in the pathogenesis and propagation of renal injury in ARF24 and there is evidence showing that administration of neutralizing anti-TNF-␣ antiserum may protect the kidney from injury.4,24,29 It is also known that TNF-␣ is a powerful cytokine with an osteoclastic stimulatory property. TNF-␣ not only activates the osteoclasts and increases osteoclastic bone resorption, it may also depress the osteoblasts, thus inhibiting bone formation.2,17,18,27
Figure 4. Osteoclast number in normal rats and in rats with acute renal failure (ARF) with or without parathyroidectomy (PTX). aP ⬍ 0.005 vs. control; bP ⬍ 0.01 vs. ARF; cP ⬍ 0.001 vs. PTX; P ⬍ 0.001 vs. control.
Bone Vol. 31, No. 4 October 2002:488 – 491
As shown by Katz et al.9 and Schneider et al.,23 TNF-␣ specifically downregulates the PTH receptors and the response to PTH in osteoblast-like cells. These investigators have also suggested that, by decreasing osteoblast sensitivity to PTH, TNF-␣ might promote an uncoupling of bone formation and resorption. It was recently suggested that TNF-␣ may induce osteoblastic apoptosis28 and that PTH may decrease apoptosis of osteoblasts.8 Thus, the reduced osteoblast number after PTX in rats with ARF (group 4) may stem from both low circulating PTH level and high TNF-␣ serum tissue level. Increased synthesis of TNF may be responsible also for the increased osteoclastic bone resorption observed in PTX rats with glycerol-induced ARF. This suggestion will need to be established by further, more detailed investigations. The present study raises the possibility that osteoclast-stimulating cytokines may play a role in bone resorption and may be involved in bone remodeling in this model of glycerol-induced ARF. Further studies are necessary, however, for the identification of factors involved in bone remodeling in other/different forms of experimental ARF as well as their correlation with PTH. References 1. Carson, F. L. and Martin, J. H. Formalin fixation for electron microscopy, a re-evaluation. Am J Clin Pathol 59:365–368; 1973. 2. Centrella, M., McCarthy, T. L., and Canalis, E. Tumor necrosis factor-␣ inhibits collagen synthesis and alkaline phosphatase activity independently of its effect on deoxyribonucleic acid synthesis in osteoblast-enriched bone cell cultures. Endocrinology 123:1442–1447; 1988. 3. Coburn, J. W., Koppel, M. H., Brickman, A. S., and Massry, S. G. Study of intestinal absorption of calcium in patients with chronic renal failure. Kidney Int 3:264 –272; 1973. 4. Fekade, D., Knox, K., Hussein, K., et al. Prevention of Jarish–Herxheimer reactions by treatment with antibodies against tumor necrosis factor ␣. N Engl J Med 335:311–315; 1996. 5. Goldner, J. A modification of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 14:237–243; 1938. 6. Goodman, W. G., Ramirez, J. A., Belin, T. R., Chon, Y., Gales, B., Segre, G. V., and Salusky, I. B. Development of adynamic bone in patients with secondary hyperparathyroidism after intermittent calcitriol therapy. Kidney Int 46:1160 –1166; 1994. 7. Hruska, K. A. and Teitelbaum, S. Renal osteodystrophy. N Engl J Med 333:166 –174; 1995. 8. Jilka, R. L., Weinstein, R. S., Bellido, T., Roberson, P., Parfitt, A. M., and Manolagas, S. C. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439 –446; 1999. 9. Katz, M. S., Gutierrez, G. E., Mundy, G. R., Hymer, T. K., Caulfield, M. P., and McKee, R. L. Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by downregulating parathyroid hormone receptors. J Cell Physiol 153:206 –213; 1992. 10. Korkor, A. B. 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. 11. Llach, F., Felsenfeld, A. J., and Haussler, M. R. The pathophysiology of altered calcium metabolism in rhabdomyolysis-induced acute renal failure. N Engl J Med 305:117–123; 1981. 12. Malluche, H. H. and Faugere, M. C. Histopathology of bone. In: Coburn, J. W., ed. Renal Osteodystrophy. den Haag, The Netherlands: Nijhoff; 1985.
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13. Malluche, H. and Faugere, M. C. Renal bone disease 1990: An unmet challenge for the nephrologist. Kidney Int 38:193–211; 1990. 14. Massry, S. G., Arieff, A. I., Coburn, J. W., Palmieri, G., and Kleeman, C. R. Divalent ion metabolism in patients with acute renal failure: Studies on the mechanism of hypocalcemia. Kidney Int 5:437–445; 1974. 15. Merke, J., Hu¨ gel, U., Zlotkowski, A., Szabo, A., Bommer, J., Mall, G., and Ritz, E. Diminished parathyroid 1,25(OH)2D3 receptors in experimental uremia. Kidney Int 32:350 –353; 1987. 16. Miller, M. A., Chin, J., Miller, S. C., and Fox, J. Disparate effects of mild, moderate, and severe secondary hyperparathyroidism on cancellous and cortical bone in rats with chronic renal insufficiency. Bone 23:257–266; 1998. 17. Miyata, T., Inagi, R., Iida, Y., Sato, M., Yamada, N., Oda, O., Maeda, K, and Seo, H. Involvement of 2-microglobulin modified with advanced glycation end products in the pathogenesis of hemodialysis-associated amyloidosis. J Clin Invest 93:521–528; 1994. 18. Owen, W. F. Jr., Hou, F. F., Stuart, R. O., Kay, J., Boyce, J., Chertow, G. M., and Schmidt, A. M. 2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts. Kidney Intern 53:1365–1373; 1998. 19. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone Histomorphometry: Standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595–610; 1987. 20. Pavlovitch, J. H., Cournot-Witmer, G., Bourdeau, A., and Balsan, S. Suppressive effects of 24,25-dihydroxycholecalciferol on bone resorption induced by acute bilateral nephrectomy in rats. J Clin Invest 68:803–810; 1981. 21. Pietrek, J., Kokot, F., and Kuska, J. Serum 25-hydroxyvitamin D and parathyroid hormone in patients with acute renal failure. Kidney Int 13:178 –185; 1978. 22. Sawaya, B. P., Koszewski, N. J., Quanle, Q. M., Langub, C., Monier-Faugere, M.-C., and Malluche, H. H. Secondary hyperparathyroidism and vitamin D receptor binding to vitamin D response elements in rats with incipient renal failure. J Am Soc Nephrol 8:271–278; 1997. 23. Schneider, H.-G., Allan, E. H., Moseley, J. M., Martin, J., and Findlay, D. M. Specific down-regulation of parathyroid hormone (PTH) receptors and responses to PTH by tumour necrosis ␣ and retinoic acid in UMR 106-06 osteoblast-like osteosarcoma cells. Biochem J 280:451–457; 1991. 24. Shulman, L. M., Yuhas, Y., Frolkis, I., Gavendo, S., Knecht, A., and Eliahou, H. E. Glycerol induced ARF in rats is mediated by tumor necrosis factor-␣. Kidney Intern 43:1397–1401; 1993. 25. Slatopolsky, E., Caglar, S., Pennell, J. P., Tagart, D. B., Canterbury, J. M., Reiss, E., and Bricker, N. S. On the pathogenesis of hyperparathyroidism in chronic experimental renal insufficiency in the dog. J Clin Invest 50:492–499; 1971. 26. Teitelbaum, S. L. Renal osteodystrophy. Hum Pathol 15:306 –323; 1984. 27. Thomson, B. M., Mundy, G. R., and Chambers, T. J. Tumor necrosis factors ␣ and  induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 138:775–779; 1987. 28. Tsuboi, M., Kawakami, A., Nakashima, T., Matsuoka, N., Urayama, S., Kawabe, Y., Fujivama, K., Kiriyama, T., Aoyagi, T., Maeda, K., and Eguchi, K. Tumor necrosis factor-alpha and interleukin-1beta increase the Fas-mediated apoptosis of human osteoblasts. J Lab Clin Med 134:222–231; 1999. 29. Wheeler, A. P. and Bernard, G. R. Treating patients with severe sepsis. N Engl J Med 340:107–214; 1999. 30. Wilson, L., Felsenfeld, A., Drezner, M. K., and Llach, F. Altered divalent ion metabolism in early renal failure: Role of 1,25(OH)2D. Kidney Int 27:565–573; 1985.
Date Received: November 22, 2001 Date Accepted: July 13, 2002
Stimulation of Osteoclastic Bone Resorption in a Model of Glycerol-Induced Acute Renal Failure: Evidence for a Parathyroid Hormone-Independent Mechanism A. GAL-MOSCOVICI, P. SCHERZER, D. RUBINGER, R. WEISS, M. DRANITZKI-ELHALEL, and M. M. POPOVTZER Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel
production rate of calcitriol,10,15,21 and a decreased calcitriol receptor concentration were recently shown in a rat model of incipient renal failure.10,22 Thus, it was shown that a reduced renal vitamin D receptor binding to the nuclear response element22 may be the underlying mechanism for vitamin D resistance in renal failure, which may lead to increased parathyroid hormone secretion. In a model of ARF due to bilateral nephrectomy Pavlovitch et al. found that serum parathyroid hormone was significantly elevated 5 h after ablation surgery.20 The bone changes found in their model of ARF included a significant increase in osteoclastic resorption. They concluded that this increase in bone resorption was partially mediated by the increased parathyroid hormone level. Whether the hypersecretion of parathyroid hormone is the only factor responsible for bone changes seen in ARF remains a matter of debate. The possibility of factors other than parathyroid hormone being involved in bone changes occuring in ARF has not been studied in detail. This study was undertaken to investigate the bone changes occurring in a model of glycerol-induced ARF and to evaluate the influence of parathyroid hormone on these changes.
This study was undertaken to evaluate the bone changes occurring in rats with acute renal failure (ARF). Acute renal failure was induced in rats 24 hours after dehydration by an intramuscular injection of glycerol. After induction of ARF, the rats were divided into two groups, one of which underwent parathyroidectomy (PTX). Rats with normal renal function, matched for age and weight, were used as controls and divided into two groups, one of them for PTX. At termination of the study blood and urine chemistry and bone histomorphometry were analyzed. Rats with glycerol-induced ARF developed bone changes compatible with mild hyperparathyroid bone disease, characterized mainly by increased osteoclastic bone resorption when compared with control rats having normal renal function. Rats with normal renal function following PTX developed bone disease showing complete suppression of forming and resorptive parameters. Rats with glycerol-induced ARF and PTX showed abolishment of all bone forming parameters, but a dramatic increase in osteoclastic resorption was apparent. Based on these observations we suggest that, in this model of glycerolinduced ARF, osteoclastic bone resorption may develop in the absence of parathyroid hormone, probably stimulated by other potent osteoclastogenic factors. (Bone 31:488 – 491; 2002) © 2002 by Elsevier Science Inc. All rights reserved.
Materials and Methods Experimental Design
Key Words: Acute renal failure (ARF); Parathyroidectomy (PTX); Rats; Osteoclastic bone resorption; Histomorphometry; Secondary hyperparathyroidism.
Male Sabra rats of the Hebrew University strain, weighing 180 –200 g, were housed in metabolic cages and allowed free access to standard pellet chow containing 0.78% calcium, 0.51% phosphorus, and tap water. Following an acclimatization period of about 1 week, the animals were divided into four groups:
Introduction Renal osteodystrophy occurs frequently in the course of chronic renal failure (CRF). Bone diseases in CRF spread over a broad spectrum of pathology ranging from hyperparathyroid to mixed and adynamic bone disease. Osteitis fibrosa cystica due to secondary hyperparathyroidism is still the most predominant form of renal osteodystrophy.6,7,12,13,26 There is evidence that secondary hyperparathyroidism also occurs in acute renal failure (ARF), even at a very early stage. An impaired calcium transport by intestine,3,11,14,30 a decreased
1. Control rats with normal renal function (sham and non-shamoperated). 2. Rats with normal renal function (sham-operated) undergoing parathyroidectomy (PTX). 3. Rats with acute renal failure (ARF). 4. Rats with ARF and PTX. ARF was induced by one intramuscular injection of 10 mg/kg body weight of 50% glycerol, following a 24 h period of dehydration. PTX was performed on the same day by hot wire cautery under light ether anesthesia. Twenty-four-hour urine collections were obtained during the last day of the experiment. Forty-eight hours after induction of
Address for correspondence and reprints: Dr. A. Gal-Moscovici, Nephrology and Hypertension Services, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. E-mail: [email protected] © 2002 by Elsevier Science Inc. All rights reserved.
488
8756-3282/02/$22.00 PII S8756-3282(02)00849-9
Bone Vol. 31, No. 4 October 2002:488 – 491
A. Gal-Moscovici et al. Parathyroid-independent osteoclastic bone resorption
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Table 1. Plasma biochemistry in control and rats with acute renal failure (ARF) and parathyroidectomy (PTX)
Control ARF PTX ARF ⫹ PTX
PCr (mol/L)
CrCl (L/min)
PCa (mEq/L)
PP (mEq/L)
PPTH (pg/mL)
46.27 ⫾ 8.4 179 ⫾ 43c 44.4 ⫾ 1.9 199 ⫾ 44d
537 ⫾ 69 125 ⫾ 39a 337 ⫾ 90 105 ⫾ 40b
4.75 ⫾ 0.09 4.58 ⫾ 0.39 2.72 ⫾ 0.24a 4.16 ⫾ 0.54d
3.5 ⫾ 0.92 3.32 ⫾ 0.38 4.53 ⫾ 0.16c 4.01 ⫾ 0.92
42.4 ⫾ 6.1 257 ⫾ 32e 13.6 ⫾ 0.67e,f 11.1 ⫾ 1.34e,f
p ⬍ 0.001 vs. control. bp ⬍ 0.05 vs. PTX. cp ⬍ 0.02 vs. control. dp ⬍ 0.01 vs. PTX. ep ⬍ 0.001 vs. control. p ⬍ 0.001 vs. ARF.
a f
ARF the animals were killed and blood samples were collected anaerobically from the abdominal aorta in 5 mL vacuum tubes for measurement of the creatinine, calcium, phosphate, and parathyroid hormone (PTH) levels. The biochemical measurements performed on an automated analyzer included determination of plasma and urine levels of creatinine, calcium, and phosphate. The PTH levels in the serum were determined by an immunoradiometric assay (IRMA) using a rat-specific PTH kit (Immutopics, Inc., San Clemente, CA). Histomorphometry Histomorphometric studies were performed on the right tibial bone. Tetracycline (Ledermycin, Lederle, Germany) at a dose of 25 mg/kg was injected intraperitoneally at 5 days and 1 day prior to killing. The duration of the study was 6 days. The tibial bones were removed, further fixed in Carson– Milloning solution,1 and embedded undecalcified in a historesin kit after dehydration in alcohol. Sections of 5 m and 10 m were cut in anter oposterior planes, using a Reichert-Jung microtome. The thicker sections (10 m) were mounted unstained for viewing of the fluorochromal marker. To identify mineralized bone, osteoid, osteoblasts, and osteoclasts, the 5 m sections were stained by the von Kossa method.5 The following parameters were calculated according to Parfitt19: 1. Osteoblast surface (Ob.S/BS), the fraction of trabecular surfaces covered by osteoblasts. 2. Osteoid surface (OS/BS), the fraction of trabecular surface covered by osteoid. 3. Osteoid volume (OV/BV), the fraction of trabecular bone volume occupied by osteoid. 4. Osteoclast surface (Oc.S/BS), the fraction of trabecular surface covered by osteoclasts. 5. Osteoclast number (Oc.N/TA), the number of osteoclasts per square millimeter of cancellous bone. 6. Osteoid thickness (O.Th), calculated as the ratio of osteoid volume to osteoid surface ⫻ 100. 7. Eroded surface (ES/BS), the fraction of trabecular surface covered by lacunae (including “active” lacunae with osteoclasts and lacunae in reversal phase; i.e., the period in which the resorption lacunae are filled with inactive-looking mononuclear cells). 8. Double-labeled tetracycline surface, the fraction of trabecular surface covered by double-labeled tetracycline. 9. Mineralized surface, the total extent of double labels plus half the extent of single labels. 10. Bone formation rate (BFR/BS) at tissue level, obtained by multiplying the mineralized surface by mineral oppositional rate. 11. Mineral appositional rate (MAR), obtained by measuring the mean distance between tetracycline labels and dividing by the number of days between doses of tetracycline.
Statistical Methods Statistical analysis of the histomorphometric data within the groups was done using nonparametric one-way analysis of variance and the SPSS statistical package. Biochemical statistical analysis was performed by the Wilcoxon rank sum test, using two-group comparison. Results are presented in the tables as mean ⫾ SE. Histomorphometry was quantified in a blinded manner by the same investigator. Random biopsy samples were assessed for verification by two different experts from Israel. The interexaminer differences were 5% for all parameters. Results Biochemical Data Rats with acute renal failure presented with increased plasma creatinine and decreased creatinine clearance when compared to rats with normal renal function (Table 1). Parathyroidectomy was documented by the presence of low calcium, high phosphate, and low PTH plasma levels. There were no changes in plasma creatinine and creatinine clearance levels in group 2 (sham-operated rats with PTX) when compared with controls (sham and non-sham). The rats with both ARF and PTX showed a decreased creatinine clearance and an increase in plasma creatinine, similar to those with ARF only. Plasma PTH level was significantly lower in the group with ARF and PTX as compared with the control group with ARF only (Table 1). Plasma calcium level in group 4 was lower than that in group 2, but the difference did not reach statistical significance. Similarly, the phosphate plasma levels in group 4 were higher than in group 2, but failed to reach statistical significance. Histomorphometric Results The histomorphometric static bone-forming parameters in the four study groups are shown in Figure 1. Rats with normal kidney function, following PTX (group 2), showed severely reduced osteoid volume and surface and absence of osteoblastic cells. These changes were statistically significant when compared with control rats or rats with ARF. In rats with ARF (group 3) a significant decrease in osteoid volume and surface, as well as in osteoblastic surface, was found when compared with control rats. The most striking suppression of osteoid volume and osteoid surface was found in the rats with both ARF and PTX (group 4). This group also showed a complete abolishment of the osteoblastic line. The dynamic bone forming parameters in groups 2, 3, and 4 showed decreased mineralizing capacity and reduced bone formation rate when compared with control rats (group 1); however, these changes were statistically nonsignificant (Figure 2). Bone-resorbing parameters of all four groups are shown in
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Figure 1. Static bone-forming parameters in control rats and in rats with acute renal failure (ARF) with and without parathyroidectomy (PTX). *P ⬍ 0.01 vs. control; **P ⬍ 0.05 vs. control; aP ⬍ 0.005 vs. PTX, ARF; b P ⬍ 0.01 vs. ARF; cP ⬍ 0.001 vs. ARF, control.
Figures 3 and 4. Rats with ARF (group 2) showed a significantly increased osteoclastic surface and osteoclast number (Figure 4) when compared with control rats (group 1). Rats with normal renal function and PTX (group 3) showed an absence of osteoclasts or osteoclastic activity. The most impressive results were observed in rats with both ARF and PTX (group 4). In this group, osteoclast number (Figure 4) and surface (Figure 3) increased 2.5-fold when compared with control rats (group 1), and was also significantly enhanced when compared to rats with ARF without PTX (group 2). Discussion The pathogenesis of renal osteodystrophy in chronic renal failure has attracted much attention. Bone disease in acute renal failure, however, has not been studied in great detail. A decrease in glomerular filtration rate (GFR) of about 40% in ARF has been reported to be associated with increased secretion of PTH.16,20,22,25 Thus, increased bone turnover with increased osteoclastic and osteoblastic activity are expected to occur. Evidence of increased osteoclastic activity, induced by bilateral nephrectomy, was reported by Pavlovitch et al. in their model of ARF, but they presented no data regarding osteoblastic activity.20 The bone abnormality in our rats with glycerol-induced ARF consisted of a significant increase in osteoclast activity, but
Figure 2. Bone formation rate in normal rats and rats with acute renal failure (ARF) with or without parathyroidectomy (PTX).
Figure 3. Osteoclastic bone resorption in normal rats and rats with acute renal failure (ARF) with or without parathyroidectomy (PTX). aP ⬍ 0.05 vs. control; bP ⬍ 0.05 vs. ARF; cP ⬍ 0.001 vs. PTX; dP ⬍ 0.001 vs. control.
differed from the classical picture of hyperparathyroid bone disease by the absence of activated osteoblasts and by the presence of depressed bone-forming parameters. Parathyroidectomy performed in these rats with ARF (group 4) showed, as expected, further reduction in osteoblastic activity. However, it was accompanied by a significant increase in osteoclastic activity when compared to rats with ARF only (group 3). This finding of increased osteoclastic number and activity in rats with ARF and PTX (group 4) suggests that factors other than PTH may be involved in osteoclast activation. The glycerol-induced model of ARF in rats is associated with an increase in endogenously produced cytokines, mainly tumor necrosis factor-␣ (TNF-␣), by activated inflammatory cells.20 –22 TNF-␣ is often implicated in the pathogenesis and propagation of renal injury in ARF24 and there is evidence showing that administration of neutralizing anti-TNF-␣ antiserum may protect the kidney from injury.4,24,29 It is also known that TNF-␣ is a powerful cytokine with an osteoclastic stimulatory property. TNF-␣ not only activates the osteoclasts and increases osteoclastic bone resorption, it may also depress the osteoblasts, thus inhibiting bone formation.2,17,18,27
Figure 4. Osteoclast number in normal rats and in rats with acute renal failure (ARF) with or without parathyroidectomy (PTX). aP ⬍ 0.005 vs. control; bP ⬍ 0.01 vs. ARF; cP ⬍ 0.001 vs. PTX; P ⬍ 0.001 vs. control.
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As shown by Katz et al.9 and Schneider et al.,23 TNF-␣ specifically downregulates the PTH receptors and the response to PTH in osteoblast-like cells. These investigators have also suggested that, by decreasing osteoblast sensitivity to PTH, TNF-␣ might promote an uncoupling of bone formation and resorption. It was recently suggested that TNF-␣ may induce osteoblastic apoptosis28 and that PTH may decrease apoptosis of osteoblasts.8 Thus, the reduced osteoblast number after PTX in rats with ARF (group 4) may stem from both low circulating PTH level and high TNF-␣ serum tissue level. Increased synthesis of TNF may be responsible also for the increased osteoclastic bone resorption observed in PTX rats with glycerol-induced ARF. This suggestion will need to be established by further, more detailed investigations. The present study raises the possibility that osteoclast-stimulating cytokines may play a role in bone resorption and may be involved in bone remodeling in this model of glycerol-induced ARF. Further studies are necessary, however, for the identification of factors involved in bone remodeling in other/different forms of experimental ARF as well as their correlation with PTH. References 1. Carson, F. L. and Martin, J. H. Formalin fixation for electron microscopy, a re-evaluation. Am J Clin Pathol 59:365–368; 1973. 2. Centrella, M., McCarthy, T. L., and Canalis, E. Tumor necrosis factor-␣ inhibits collagen synthesis and alkaline phosphatase activity independently of its effect on deoxyribonucleic acid synthesis in osteoblast-enriched bone cell cultures. Endocrinology 123:1442–1447; 1988. 3. Coburn, J. W., Koppel, M. H., Brickman, A. S., and Massry, S. G. Study of intestinal absorption of calcium in patients with chronic renal failure. Kidney Int 3:264 –272; 1973. 4. Fekade, D., Knox, K., Hussein, K., et al. Prevention of Jarish–Herxheimer reactions by treatment with antibodies against tumor necrosis factor ␣. N Engl J Med 335:311–315; 1996. 5. Goldner, J. A modification of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 14:237–243; 1938. 6. Goodman, W. G., Ramirez, J. A., Belin, T. R., Chon, Y., Gales, B., Segre, G. V., and Salusky, I. B. Development of adynamic bone in patients with secondary hyperparathyroidism after intermittent calcitriol therapy. Kidney Int 46:1160 –1166; 1994. 7. Hruska, K. A. and Teitelbaum, S. Renal osteodystrophy. N Engl J Med 333:166 –174; 1995. 8. Jilka, R. L., Weinstein, R. S., Bellido, T., Roberson, P., Parfitt, A. M., and Manolagas, S. C. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439 –446; 1999. 9. Katz, M. S., Gutierrez, G. E., Mundy, G. R., Hymer, T. K., Caulfield, M. P., and McKee, R. L. Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by downregulating parathyroid hormone receptors. J Cell Physiol 153:206 –213; 1992. 10. Korkor, A. B. 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. 11. Llach, F., Felsenfeld, A. J., and Haussler, M. R. The pathophysiology of altered calcium metabolism in rhabdomyolysis-induced acute renal failure. N Engl J Med 305:117–123; 1981. 12. Malluche, H. H. and Faugere, M. C. Histopathology of bone. In: Coburn, J. W., ed. Renal Osteodystrophy. den Haag, The Netherlands: Nijhoff; 1985.
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Date Received: November 22, 2001 Date Accepted: July 13, 2002