Effect of aluminum and parathyroid hormone on osteoblasts and bone mineralization in chronic renal failure

Summary Bone aluminum, quantitative bone histology, and plasma parathyroid hormone (PTH) were compared in 29 patients undergoing chronic hemodialysis. Histologic techniques included double tetracycline labeling and histochemical identification of osteoclasts and osteoblasts. Bone aluminum was measured chemically by flameless atomic absorption spectrophotometry, and histochemically. When measured chemically, the bone aluminum was 67±46 (SD) mg/kg dry weight (normal 2.4±1.2 mg/kg); histochemically, aluminum was present at 2.9±4.4% of trabecular surface. The biochemical and histochemical results agreed well (r=0.80,P<0.001). No double tetracycline labels were seen at the mineralization front where aluminum was deposited, indicating cessation of mineralization at these sites. The osteoblast surface correlated positively with plasma PTH (r=0.67,P<0.001) and negatively with bone aluminum level (r=−0.42,P<0.05). Multiple linear regression showed a correlation of aluminum with osteoblasts additional to that of PTH, consistent with a direct effect of aluminum in depressing osteoblast numbers. Though a relationship between PTH and chemically determined bone aluminum level could not be demonstrated, there was a negative correlation between osteoclast count and aluminum, and the nine patients with severe hyperparathyroid bone disease had lower chemically determined aluminum levels than the other patients. These results suggest that aluminum (a) directly inhibits mineralization, (b) is associated with decreased PTH activity and hence osteoblast numbers, and (c) directly reduces osteoblast numbers. In addition to inducing severe, resistant osteomalacia, aluminum appears to contribute to the mild osteomalacia commonly seen in renal failure, characterized by extensive thin osteoid and low tetracycline and osteoblast surfaces.     

Aluminum administration in the rat separately affects the osteoblast and bone mineralization

Aluminum administration in the experimental animal results in osteomalacia as characterized by osteoid accumulation and decreased mineralization. Previous in vivo and in vitro studies have indicated that either aluminum directly inhibits mineralization or is toxic to the osteoblast. In the present study, PTH was continuously infused in rats with aluminum-induced osteomalacia to evaluate whether aluminum administration decreased mineralization without a concomitant decrease in osteoblasts. Four groups of rats were studied: chronic renal failure (CRF); CRF + aluminum (AL); CRF + PTH; and CRF + PTH + AL. Rats were sacrificed 5 and 12 days after aluminum or diluent administration; in the PTH groups, bovine PTH (1-34) was administered at 2 units/h via a subcutaneously implanted Alzet pump. Aluminum administration decreased osteoblast surface, increased osteoid accumulation, and produced a cessation of bone formation. The infusion of PTH alone increased osteoblast surface and bone formation. The simultaneous administration of aluminum and PTH resulted in an osteoblast surface intermediate between aluminum and PTH alone; however, despite a PTH-induced restoration of osteoblast surface, bone formation did not increase. These findings indicate (1) aluminum is toxic to osteoblasts and also directly inhibits mineralization even when osteoblasts are not decreased; (2) PTH is capable of increasing osteoblasts even in the presence of aluminum; and (3) despite a PTH-induced increase in osteoblast surface, mineralization of osteoid was not improved.     

Parenteral aluminum administration in the dog: II. Induction of osteomalacia and effect on vitamin D metabolism

There is an association between bone aluminum (Al) accumulation and dialysis-associated osteomalacia (OM). To study whether Al is pathogenic in OM, quantitative bone histomorphometry was done in six dogs before (Bx 1) and after (Bx 2) 3 to 5 weeks of intravenous Al administration (1 mg Al /kg/day). Bone Al was determined by histochemical and chemical methods. The percent osteoid rose from 2.8 +/- 0.8 to 7.0 +/- 4.3% (mean +/- SD), P less than 0.05, and osteoid width increased from 5.7 +/- 0.6 to 8.0 +/- 1.2 mu, P less than 0.01, after Al. Bone Al rose from 1.3 +/- 1.6 to 94.0 +/- 19.0 mg/kg after Al, and the severity of OM, expressed as either percent forming surface or percent osteoid, correlated with bone Al measured histochemically and expressed as either percent surface or percent area of trabecular bone staining for Al (r = 0.85 - 0.90, P less than 0.01). Poor tetracycline uptake (six dogs), which indicates impaired mineralization, and little or no separation of tetracycline labels (four dogs) were noted at Bx 2; thus, bone apposition and formation rates were below the limits of detection. Resorptive surface did not change but trabecular volume, expressed as percent of tissue volume, fell from 22.1 +/- 3.0 to 17.1 +/- 1.4%, P less than 0.05. Serum levels of 1,25(OH)2D fell from 26.8 +/- 9.1 to 4.5 +/- 5.5 pg/ml after 17 days of Al; serum 25(OH)D levels were unchanged. These data indicate that Al can cause OM and that its severity correlates with the bone Al content.2 +     

The evolution of osteomalacia in the rat with acute aluminum toxicity

Aluminum toxicity is the presumed cause of aluminum-associated osteomalacia. In animal models, osteomalacia has been produced after a prolonged course of aluminum. In the present study, rats with renal failure received 20 mg intraperitoneal aluminum during a 2 day period. This model allows sequential observations in the development of osteomalacia. Rats were sacrificed and studied 5, 12, 25, and 40 days after aluminum administration. No differences were observed in serum calcium, phosphorus, or creatinine as a consequence of aluminum administration. Compared with control rats, parathyroid hormone was decreased at 12 and 25 days. A direct correlation was present between plasma and bone aluminum at 12 days (r = 0.92, p less than 0.01), 25 days (r = 0.85, p less than 0.005), and 40 days (r = 0.88, p less than 0.001) but not 5 days after aluminum administration. Plasma aluminum peaked at 5 days (727 +/- 89 micrograms/liter, mean +/- SEM) and bone aluminum at 40 days (273 +/- 40 micrograms/g). Aluminum had profound effect on bone histology. At 5 days there was a decrease in osteoblast surface and osteoid surface; at 12 days osteoblast surface and osteoid surface returned to normal but osteoclast surface decreased. Subsequently there was a progressive increase in osteoid surface and osteoid volume. Bone formation rate measured at 12, 25, and 40 days was decreased at these intervals. In conclusion, (1) high plasma aluminum may be directly toxic to the osteoblast; (2) progressive osteoid accumulation is secondary to matrix (osteoid) deposition, which exceeds the depressed bone formation rate; (3) the progressive decrease in plasma aluminum and increase in bone aluminum suggest that bone has a high affinity for aluminum but may have a relatively slow rate of uptake at any given time; (4) aluminum may directly decrease parathyroid hormone; (5) the correlation between plasma and bone aluminum suggest an exchange is present; and (6) aluminum toxicity may independently affect the osteoblast and bone mineralization.     

The effect of high parathyroid hormone levels on the development of aluminum-induced osteomalacia in the rat

A relative deficiency of parathyroid hormone (PTH) is generally observed in dialysis patients with aluminum-associated osteomalacia or aplastic bone disease. It has been suggested that high PTH levels may protect against the development of aluminum-associated bone disease. Through the use of a previously established model of aluminum-induced osteomalacia in the rat, the protective effect of PTH was evaluated. Aluminum was administered intraperitoneally at doses of 0, 5, 10, and 20 mg during a 2-day period, and rats were sacrificed 5 and 12 days after aluminum administration. PTH (bovine 1-34) was administered via a subcutaneously implanted Alzet pump at 2 U/h starting 4 days before aluminum administration and continuing until sacrifice. As the aluminum dose was increased to 20 mg, the osteoblast surface and the bone formation rate decreased. PTH supplementation increased the osteoblast surface at all doses of aluminum and increased the bone formation rate at 0 and 5 mg of aluminum. However, even with PTH supplementation, osteoblast surface decreased as the aluminum dose increased. In the absence of PTH supplementation, osteoblast surface was markedly reduced when the serum aluminum concentration was greater than 400 micrograms/liter or stainable trabecular aluminum surface exceeded 15%. When the stainable trabecular aluminum surface was greater than 12%, the bone formation rate was zero even during supplemental PTH administration. A significant correlation was observed between serum aluminum and stainable trabecular aluminum surface (r = 0.80 at 5 days and r = 0.86 at 12 days; P less than 0.001). However, after PTH administration, less stainable trabecular aluminum was present for the same serum aluminum concentration. Both with and without PTH, the slope of the correlation between serum aluminum and stainable trabecular aluminum surface was steeper at 5 days after aluminum administration than at 12 days. In conclusion, for an equivalent aluminum exposure, high PTH levels protected against the development of low turnover aluminum bone disease in the rat.     

Differences in bone turnover and intact PTH levels between African American and Caucasian patients with end-stage renal disease

BACKGROUND: Evidence derived from healthy subjects suggests that African Americans have higher serum parathyroid hormone (PTH) levels and decreased bone responsiveness to PTH than Caucasians. African American patients with end-stage renal disease (ESRD) also have higher serum PTH than Caucasians. Studies that correlate intact PTH (iPTH) levels with bone turnover in ESRD patients were performed in a predominantly Caucasian population. METHODS: In this study, serum iPTH and bone histomorphometric data were analyzed for racial differences in 76 ESRD patients (Caucasian = 48, African Americans = 28). Bone turnover was determined by histomorphometric measurement of activation frequency in all patients. RESULTS: Age, duration of dialysis, and calcium and phosphorus levels were similar between the two groups. iPTH levels (pg/mL; mean +/- SE) were significantly higher in the African American group (534 +/- 79 vs. 270 +/- 46, P < 0.01). Also, alkaline phosphatase levels (IU/L) were significantly higher in the African American group (162 +/- 31 vs. 144 +/- 43, P < 0.01). Correlations between PTH levels and activation frequency were r = 0.60, P < 0.01 in Caucasians and r = 0.22, P = NS in African Americans. The mean PTH level in African American patients with histologic findings of low bone turnover was 460 +/- 115 vs. 168 +/- 41 in Caucasian patients with similar bone turnover (P < 0.01). In patients with low bone turnover, African Americans had significantly higher osteoid volume and thickness, number of osteoblasts and osteoclasts, erosion surface, peritrabecular fibrosis, and single-label surface than Caucasians. However, erosion depth, bone formation rate per osteoblast and mineralization apposition rate were similar between the two groups. CONCLUSION: There is no correlation between iPTH and bone turnover in African Americans with ESRD. A substantial number of African American patients with low bone turnover have very high serum PTH levels. Bone histomorphometric results reveal differences in remodeling dynamics and responses to PTH between African American and Caucasian patients. Further studies utilizing newer PTH measurement assays are needed to better delineate the correlation between PTH and bone turnover in the various racial groups.     

Effect of parathyroidectomy on aluminum toxicity and azotemic bone disease in the rat

In maintenance dialysis patients, low-turnover osteomalacia and aplastic bone disease are generally attributed to aluminum toxicity. Both groups of patients have a relative deficiency of PTH. The reason for the development of osteomalacia versus aplastic bone disease is unclear. The present study was performed to evaluate whether parathyroidectomy (PTX) modifies the effect of aluminum administration on bone histology in renal failure. Seven groups of pair-fed rats were studied: normals (N); renal failure (RF); RF + PTX; PTX; RF + aluminum (AL); RF + PTX + AL; and PTX + AL. Aluminum was administered intraperitoneally 5 days/week for 6 weeks. All groups were sacrificed at 6 weeks. Renal failure increased the serum calcium in both the parathyroid intact (RF versus N, 11 +/- 0.1 versus 10 +/- 0.3 mg/dl, X +/- SEM, P less than 0.05) and calcium-supplemented PTX groups (PTX + RF versus PTX, 9.7 +/- 0.2 versus 9.2 +/- 0.2 mg/dl, P less than 0.05). After PTX, aluminum administration increased the serum calcium (PTX + AL versus PTX, 9.8 +/- 0.3 versus 9.2 +/- 0.2, P less than 0.05, and PTX + RF + AL versus PTX + RF, 10.8 +/- 0.1 versus 9.7 +/- 0.2 mg/dl, P less than 0.05). In rats with renal failure receiving aluminum, PTX decreased osteoid volume and surface but not osteoid thickness. Rats receiving aluminum did not mineralize bone. Additionally, in PTX rats receiving aluminum, renal failure per se increased osteoblast surface, osteoid surface, osteoid volume, and osteoclast number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortical v trabecular bone aluminum in dialyzed patients

Differences among measurement of cortical and trabecular bone aluminum (AI) have been observed. Furthermore, its relationship to bone histology has been variable. In order to clarify these points, we have evaluated measurements of bone AI in relation to the source of AI and bone lesion in 25 hemodialysis patients. All patients were dialyzed in the same unit since commencement of dialysis and treated by the same physician. Age of the patients ranged from 29 to 66 years; mean duration of dialysis was 6.6 +/- 3.5 years. Dialysate water has been treated by reverse osmosis since 1980. Bone biopsy was performed in all patients after double tetracycline labeling. AI was measured biochemically in cortical bone (bCAI) and histochemically in trabecular (TAI) and cortical bone (CAI). Mean serum AI (36 +/- 21 micrograms/L) and bCAI (59 +/- 44 micrograms/g) were increased. There were significant correlations between: cortical AI and (1) serum AI (r = 0.71, p less than 0.001); (2) duration of dialysis with softened water (AI content, 55 +/- 21 micrograms/L, r = 0.65, P less than 0.001) but not with total duration of dialysis; and (3) AI ingested since commencement of dialysis (r = 0.57, P less than 0.01). Trabecular AI was not correlated with any of these parameters. None of cortical AI measurements were correlated with bone formation rates (BFR), osteoblastic surfaces (ObS), and resorption surfaces (RS) determined on trabecular bone. However, trabecular AI was inversely correlated with BFR (P less than 0.01) and ObS (P less than 0.05). Serum parathyroid hormone (PTH) was positively correlated with BFR (P less than 0.001) and RS (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and histologic features of iron-related bone disease in dialysis patients

Forty-eight dialysis patients undergoing bone biopsy were analyzed for clinical history, blood biochemical values, bone histologic findings, bone aluminum content (BAC), bone iron content (BIC), bone iron stores, and histochemical staining of bone aluminum and bone iron. Four patients had significant trabecular bone iron staining alone; eight patients had significant bone iron and bone aluminum staining; 13 patients had significant bone aluminum staining alone; and 23 patients showed no significant bone aluminum or iron staining. Patients with significant bone iron staining were younger (37.4 +/- 5.3 years v 53.2 +/- 2.3 years, P less than 0.01, mean +/- SEM) and were more likely to be anephric (P less than 0.001) and to have a history of prior renal transplantation (P less than 0.10). The 12 patients with significant bone iron staining had received more blood transfusions than those without bone iron staining (96 +/- 22.8 U v 22 +/- 5.8 U, P less than 0.005). Patients with bone iron accumulation had higher levels of serum ferritin (3,594 +/- 1,138.4 micrograms/L [ng/mL] v 265 +/- 60.1 micrograms/L, P less than 0.01) and lower levels of immunoreactive parathyroid hormone (iPTH) (349 +/- 150 microLEq/mL v 1,801 +/- 397 microLEq/mL [386 +/- 166 pmol/L v 1,990 +/- 439 pmol/L], P less than 0.005). BIC was also higher in these patients (1,008 +/- 149 micrograms iron/g bone v 300 +/- 46.5 micrograms iron/g bone, P less than 0.001) and higher than normal BIC (256 +/- 44.2 micrograms iron/g bone, eight normals). Bone marrow iron stores were positively related to serum ferritin levels (P less than 0.01) and trabecular bone iron staining (P less than 0.10). All 13 patients with osteomalacia demonstrated significant bone aluminum staining; seven of these patients demonstrated concomitant significant iron staining. Fourteen of 15 patients with severe hyperparathyroidism showed no significant iron or aluminum staining. Our data indicate that iron will probably not accumulate within bone until all other storage sites (eg, bone marrow) are fully saturated. The presence of lower levels of iPTH in iron-overloaded patients raises the possibility that iron overload may induce a state of relative hypoparathyroidism. The most important determinant for the presence of osteomalacia seems to be the presence of significant aluminum staining. No specific bone histologic finding was related to the presence of bone iron staining, but the rarity of isolated significant bone iron staining makes it difficult to evaluate bone histologic diagnoses that might be solely attributable to iron.     

Stainable aluminum and not aluminum content reflects bone histology in dialyzed patients

Quantitative evaluation of stainable bone aluminum and measurement of bone aluminum content were done in 55 patients on chronic maintenance dialysis. All patients underwent bone biopsies. Histomorphometry of static and dynamic parameters of bone structure, bone formation and resorption, and quantitation of stainable bone aluminum at the osteoid-bone interface were performed. In addition, bone aluminum content was measured by atomic absorption spectrophotometry. Bone aluminum content was elevated in all patients (81 +/- 9.6 vs. 18 +/- 6 micrograms/g dry wt) and stainable aluminum was found in 47% of them. All patients with predominant low-turnover osteomalacia or adynamic bone disease displayed stainable bone aluminum. In contrast, stainable bone aluminum was not present in individuals with predominant-hyperparathyroid bone disease. Patients with stainable aluminum had lower bone mass (P less than 0.05), higher volume and surface of lamellar osteoid (P less than 0.01), less volume and surface of woven osteoid (P less than 0.05 and P less than 0.01), lower osteoblastic and osteoclastic indices (P less than 0.01), less doubly labelled osteoid seams, lower mineral apposition rate and lower bone formation rates (P less than 0.05 to P less than 0.01). Stainable aluminum correlated with volume of lamellar osteoid and cellular parameters of bone formation and resorption, mineral apposition rate, and bone formation rates (P less than 0.05 to P less than 0.001). In contrast, bone aluminum content correlated with volume of lamellar osteoid only (P less than 0.001). These findings indicate that stainable aluminum at the mineralization front and not aluminum content of bone reflects the histopathologic changes found in bone of dialyzed patients.     

Effect of fluoride on aluminum-induced bone disease in rats with renal failure.

Aluminum (Al) accumulation in renal failure is an etiological factor in the pathogenesis of low turnover bone disease. Aluminum-induced impairment of mineralization has been related to a reduced extent of active bone-forming surface. The present study investigated the effect of fluoride, a potent stimulator of osteoblast number, on the toxicity of aluminum in rats with renal failure (Nx). Following a large parenteral aluminum load (3.2 mg/kg x day) over a period of nine weeks, bone histomorphometry of vertebral cancellous bone revealed a severe low-turnover osteodystrophy as evidenced by a fall in osteoblastic osteoid surfaces and mineral apposition rates. Concurrent administration of fluoride [20 mg/liter (F20) or 40 mg/liter (F40) supplied with the drinking water] resulted in a significant increase in the number of osteoblasts (Nx+Al+F40 vs. Nx+Al, 33.75 +/- 2.83 vs. 1.81 +/- 0.43 mm-1, P less than 0.001) together with an overall reduced deposition of aluminum in bone (469.3 +/- 24.6 vs. 592.2 +/- 28.3 micrograms/g, P less than 0.01). However, there was an increase in the fraction of osteoid surface exhibiting stainable aluminum at the bone-osteoid interface (70.7 +/- 7.1 vs. 44.3 +/- 6.0%, P less than 0.005). Fluoride-exposed rats accumulated a significantly larger osteoid volume, suggesting an exacerbation of the osteomalacic lesion, and furthermore, dynamic histomorphometric parameters remained depressed. These results indicate that fluoride has a distinct effect on the pattern of aluminum deposition in bone. In addition, fluoride antagonizes the aluminum-induced reduction in osteoblast number but provides no amelioration of the impaired mineralization in aluminum-intoxicated rats. Thus, in this model a decrease in the extent of osteoblast surface does not account for the development of aluminum-related bone disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

High prevalence of low bone turnover and occurrence of osteomalacia after kidney transplantation

Kidney transplantation corrects most of the metabolic abnormalities that cause renal osteodystrophy. However, many transplanted patients develop osteoporosis and other bone lesions that are related, at least in part, to their immunosuppressive regimen. The precise histologic patterns of bone disease after transplantation are not well defined. In a study designed to investigate this issue, 57 adult posttransplant patients agreed to undergo bone biopsies and blood drawings. There were 32 men and 25 women, mean age 45 +/- 2 yr, who had received a kidney transplantation 5.6 +/- 0.8 yr before biopsy. History of bone pain, fractures, and avascular necrosis was found in 22, 12, and 7 patients, respectively. Serum creatinine was 1.68 +/- 0.1 mg/dl, 21% of patients were hypercalcemic, 63.2% had elevated parathyroid hormone (PTH) (>65 pg/ml), and 91.2% had normal calcitriol levels. Cancellous bone volume/tissue volume was below normal compared to age- and gender-matched control subjects in 56.1% of patients. Bone turnover (activation frequency) was low in 45.6%, normal in 28.1%, and elevated in 26.3% of patients. Bone formation rate/bone surface was low in 59.7%, normal in 35%, and elevated in 5. 3% of the patients. Erosion surface/bone surface was high in 21.1% of patients. Mineralization was prolonged in 87.5% of patients, including 9 patients with osteomalacia and 12 patients with focal osteomalacia. Cumulative and maintenance doses of prednisone and time elapsed since transplantation correlated negatively with bone volume and bone turnover (r = -0.32 to -0.59, P < 0.05 to 0.01), whereas cumulative doses of cyclosporine or azathioprine, age, gender, or serum PTH levels did not. Regression analysis identified prednisone as the main factor responsible for low bone volume and bone turnover (r = 0.54 and r = 0.43, P < 0.01). No factors were found to predict delayed mineralization. The present study shows that low bone volume, low bone turnover, and generalized or focal osteomalacia are frequent histologic features in transplanted patients. The effects of age, gender, PTH, and cyclosporine on bone volume and bone turnover are apparently overridden by the prominent effects of glucocorticoids. The prevalence of mineralization defect in the presence of normal serum levels of calcidiol and calcitriol suggests vitamin D resistance and deserves further study.     

Bone histomorphometry of renal osteodystrophy in diabetic patients

Bone biopsies and plasma parathyroid hormone (PTH) from 27 diabetic dialysis patients were compared to biopsies and PTH levels from matched patients without diabetes to determine if PTH has a role in preserving bone mass in diabetic renal osteodystrophy. Significantly lower values were present in the diabetic group for mineralized bone area (p less than 0.003), osteoblastic osteoid (p less than 0.01), resorptive surface (p less than 0.001), fibrosis (p less than 0.005), bone apposition rate (p less than 0.01), bone formation rate (BMU level) (p less than 0.04), and plasma PTH (p less than 0.05). Bone-surface aluminum was higher in the diabetic group (44 +/- 5% vs. 20 +/- 5%, p less than 0.005). Linear regression analysis revealed significant positive correlations of mineralized bone area with time on dialysis, bone formation rate, bone resorption, and PTH only in the group without diabetes. While both groups had significant positive correlations of PTH with osteoblastic osteoid and bone resorption, only in the nondiabetic group was there a positive correlation of PTH with bone apposition and bone formation rate (BMU level), observations suggesting that the lower bone formation in the diabetic patients may have arisen in part from a failure of PTH to promote bone mineralization. We conclude that relatively low PTH levels and high bone aluminum in diabetic patients with chronic renal failure may be responsible in part for low bone mass when compared to uremic patients without diabetes.     

Downregulation of osteoblast Phex expression by PTH

Human/murine X-linked hypophosphatemia is a dominant disorder associated with renal phosphate wasting and defective bone mineralization. This disorder results from mutations in the PHEX/Phex (Phosphate-regulating gene with homologies to endopeptidases on the X chromosome) gene, which is expressed in fully differentiated osteoblasts. The purpose of the present study was to assess whether PTH, a major regulator of bone development and turnover, modulates osteoblastic Phex expression. The effects of different concentrations of PTH (rat fragment 1-34) were determined on Phex mRNA and protein expression in vitro using MC3T3-E1 osteoblastic cells and mouse primary osteoblasts; and in vivo using 45-day-old mice infused for 3 days with PTH. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relative to GAPDH mRNA levels. Phex protein levels were analyzed by immunoprecipitation of 35S-methionine-labeled osteoblast lysates or by immunoblotting of calvaria membrane extracts using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide. Fully differentiated MC3T3-E1 cells were incubated for 4 to 48 h with increasing concentrations of PTH (10(-11) to 10(-7) M). PTH inhibited Phex mRNA expression in both mineralizing and nonmineralizing osteoblast cultures in a dose- and time-dependent manner with a maximal inhibition at 10(-7) M PTH after 24 h (15+/-7% of control levels, n=5, P<0.001). The PTH-mediated downregulation of Phex mRNA levels was associated with corresponding decreases in Phex protein synthesis and suppression at 10(-7) M PTH. Similar results were obtained with primary osteoblasts isolated from newborn mouse calvaria. Consistent with the in vitro findings, continuous PTH infusion to mice elicited decreases in Phex expression in calvaria. The effect of PTH was also assessed on matrix mineralization by mature MC3T3-E1 cells by measuring 45Ca accumulation in cell layers. PTH (10(-7) M) inhibited the initiation (57+/-2% of control levels, n=5, P<0.001) and the progression of matrix mineralization (75+/-1% of control levels, n=5, P<0.001). In summary, PTH inhibits osteoblastic Phex expression in vitro and in vivo. The downregulation of Phex expression by PTH in vitro is associated with inhibition of matrix mineralization, consistent with a role for Phex in bone mineralization.     

The pathogenesis of osteodystrophy after renal transplantation as detected by early alterations in bone remodeling

BACKGROUND: Loss of bone mass after transplantation begins in the early periods after transplantations and may persist for several years, even in patients with normal renal function. While the pathogenesis of these abnormalities is still unclear, several studies suggest that preexisting bone disease, glucocorticoid therapy, and alterations in phosphate metabolism may play important roles. Recent studies indicate that osteoblast apoptosis and impaired osteoblastogenesis play important roles in the pathogenesis of glucocorticoid-induced osteoporosis. OBJECTIVES: To examine the early alterations in osteoblast number and surfaces during the period following renal transplantation. METHODS: Twenty patients with a mean age of 36.5 +/- 12 years were subjected to bone biopsy 22 to 160 days after renal transplantation. In 12 patients, a control biopsy was performed on the day of transplantation. Bone sections were evaluated by histomorphometric analysis and cell DNA fragmentation by the methods of terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL), using immunoperoxidase and direct immunofluorescence techniques. RESULTS: The main alterations in posttransplant biopsies were a decrease in osteoid and osteoblast surfaces, adjusted bone formation rate, and prolonged mineralization lag time. Peritrabecular fibrosis was markedly decreased. None of the pretransplant biopsies revealed osteoblast apoptosis. In contrast, TUNEL-positive cells in the proximity of osteoid seams or in the medullary space were observed in nine posttransplant biopsies of which four had mixed bone disease, two had adynamic bone disease, one had osteomalacia, one had osteitis fibrosa, and one had mild hyperparathyroid bone disease. Osteoblast number in posttransplant biopsies with apoptosis was lower as compared with posttransplant biopsies without apoptosis. In addition, most of them showed a marked shift toward quiescence from the cuboidal morphology of active osteoblasts. Serum phosphorus levels were lower in patients showing osteoblast apoptosis and correlated positively with osteoblast number and negatively with the number of apoptotic osteoblasts. In addition, posttransplant osteoblast surface correlated positively with parathyroid hormone (PTH) levels and negatively with glucocorticoid cumulative dose. CONCLUSION: The data suggest that impaired osteoblastogenesis and early osteoblast apoptosis may play important roles in the pathogenesis of posttransplant osteoporosis. The possible mechanisms involved in the pathogenesis of theses alterations include posttransplant hypophosphatemia, the use of glucocorticoids, and the preexisting bone disease. PTH seems to have a protective effect by preserving osteoblast survival.     

Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.     

The effects of discontinuation of aluminum exposure on aluminum-induced osteomalacia

Studies in patients on dialysis have shown that aluminum (Al) accumulation in bone plays a major role in the pathogenesis of osteomalacia. It has been suggested that deferoxamine (DFO) may be beneficial in the treatment of aluminum-induced osteomalacia. The present studies were performed in four groups of uremic rats to determine if DFO and/or discontinuation of Al administration have an effect on bone histomorphometry and blood chemistries. The groups were: 1) uremic control 2) aluminum (0.75 to 1.0 mg/rat i.p., five times a week for twelve weeks): 3) aluminum + DFO, after twelve weeks Al was discontinued and the rats received DFO (75 mg/rat two times a week for nine weeks); 4) aluminum + time, after twelve weeks Al was discontinued and the rats were sacrificed after nine weeks. High levels of Al in serum and bone and low levels of PTH were seen in rats receiving Al. Bone histology revealed Al at the mineralization front, abnormal tetracycline uptake, and an increase in osteoid. DFO treatment did not significantly change the level of Al in bone, however both DFO treatment and discontinuation of Al reversed towards normal the above described lesions. In conclusion, these studies suggest that DFO and/or discontinuation of Al administration to rats with approximately 30% of renal function greatly improve aluminum-induced osteomalacia.     

Elevated bone aluminum and suppressed parathyroid hormone levels in hypercalcemic dialysis patients

We studied 21 dialysis patients who became hypercalcemic without vitamin D or calcium therapy and compared them to 28 dialysis patients who were not hypercalcemic. In the hypercalcemic group, the mean ionized-calcium level was elevated compared to normal subjects (5.4 +/- 0.4 vs. 4.9 +/- 0.1; p less than 0.001), while the ionized-calcium level in the control dialysis patients was below normal (4.5 +/- 0.4 vs. 4.9 +/- 0.1; p less than 0.001). Bone biopsies were performed in all patients. Two thirds of the hypercalcemic patients had low-turnover osteodystrophy (LTO, predominantly osteomalacia), a fraction significantly higher than in the control dialysis patients (13/21 vs. 8/28, respectively; p less than 0.05). The hypercalcemic patients with LTO had markedly elevated surface bone aluminum (63 +/- 24% of all trabecular surfaces). In contrast, the nonhypercalcemic dialysis patients with LTO and all patients with osteitis fibrosa had minimal surface bone aluminum. Hypercalcemic patients with osteitis fibrosa had a significantly lower mean N-terminal parathyroid hormone (PTH) value than did nonhypercalcemic patients with osteitis fibrosa (149 +/- 81 vs. 278 +/- 135 pg/ml, respectively; p less than 0.005). Both mean values were markedly elevated in comparison with those obtained in normal subjects (16 +/- 5 pg/ml). In contrast, patients with LTO, irrespective of the calcium level, had mean PTH values that were not significantly different from those of normal subjects. A PTH level greater than 100 pg/ml was 95% sensitive and 87% specific for osteitis fibrosa, as demonstrated by histomorphometry in nonhypercalcemic dialysis patients. However, this level was only 62% sensitive and 77% specific for a diagnosis of osteitis fibrosa in hypercalcemic dialysis patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histomorphometric evidence of deleterious effect of aluminum on osteoblasts

Bone histomorphometry was performed in 26 hemodialyzed patients to study the relation between the dynamic parameters of bone formation and aluminum deposition. Patients were divided into two groups according to whether bone formation rate at tissue level (Svft) was above or below normal: 0.089 mu 3/mu 2 per day. The 12 patients who constituted group II, defined by a Svft less than 0.089 mu 3/mu 2 per day, had markedly decreased extent of double-labeled surfaces (m = 1.3 +/- 6.5%), and these were absent in 8 of 12 patients. Osteomalacia, defined by decreased formation with increased mean osteoid thickness (greater than 15 micron), was present in only 3 of 12 patients in group II. The 14 patients who constituted group I, defined by a Svft greater than 0.089 mu 3/mu 2 per day, had both increased total labeled surfaces and mineralization rate. Osteomalacia was present in none of the group I patients. In trabecular bone, group II patients had increased stainable aluminum deposition, compared to group I patients, whether estimated as total stainable aluminum (2.16 +/- 1.34 vs 0.17 +/- 0.28 mm/mm2) or stainable percent of trabecular surfaces (42 +/- 19 vs 4 +/- 5%). This last parameter was inversely related to osteoblastic surfaces (r = -0.49, n = 26, P less than 0.01) and total labeled surfaces (r = -0.72, n = 26, P less than 0.01). Therefore, massive aluminum deposition was not invariably associated with impaired mineralization but with decreased formation due to decreased extent of active formation surfaces. In the group I patients, moderate aluminum deposition was not associated with the mineralization arrest observed in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal fluorosis: histomorphometric analysis of bone changes and bone fluoride content in 29 patients

Bone fluoride content (BFC) was measured and histomorphometric analysis of undecalcified sections was performed in transiliac biopsy cores from 29 patients (16 men, 13 women, aged 51 +/- 17 years) suffering from skeletal fluorosis due to chronic exposure to fluoride. The origin of the exposure, known in 20 patients, was either hydric (endemic or sporadic) or industrial, or in a few cases iatrogenic. Measured on calcined bone using a specific ion electrode, BFC was significantly high in each specimen (mean +/- SD; 0.79 +/- 0.36% on bone ash). The radiologically evident osteosclerosis observed in each patient was confirmed by a significant increase in cancellous bone volume (40.1 +/- 11.2% vs. 19.0 +/- 2.8% in controls, p less than 0.0001). There were significant increases in cortical width (1292 +/- 395 mcm vs. 934 +/- 173 mcm, p less than 0.0001) and porosity (14.4 +/- 6.4% vs. 6.5 +/- 1.7%, p less than 0.002), but without reduction of cortical bone mass. Cancellous osteoid volume and perimeter, as well as width of osteoid seams, were significantly increased in fluorotic patients. The increase in cancellous osteoid perimeter was almost three-fold greater than that noted in cancellous eroded perimeter. In 15 patients doubly labeled with tetracycline, the mineral apposition rate was significantly decreased, mineralization lag time was significantly increased. The fluorotic group had a greater number of osteoblasts than controls with a very high proportion of flat osteoblasts. The ultrastructural characteristics reflecting the activity of the bone cells were clearly visible on electron microscopy. Bone formation rate and adjusted apposition rate were significantly decreased in skeletal fluorosis. On stained sections and microradiographs, bone tissue showed typical modifications for skeletal fluorosis (linear formation defects, mottled bone). The volume of cancellous interstitial mineralization defects and the proportion of mottled periosteocytic lacunae were markedly increased in skeletal fluorosis. These two parameters were significantly correlated together but neither of these was significantly correlated with BFC. Renal function did not significantly influence the changes in BFC and histomorphometry of fluorotic patients. Skeletal fluorosis is thus characterized by an unbalanced coupling in favor of bone formation, and a great number of osteoblasts with a high proportion of flat osteoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)