Effect of cyclosporin A on bone mineral metabolism in experimental diabetes mellitus in the rat

JOURNAL OF BONE AND MINERAL RESEARCH Volume 9, Number 4, 1994 Mary Ann Liebert, Inc., Publishers

Effect of Cyclosporin A on Bone Mineral Metabolism in Experimental Diabetes Mellitus in the Rat

S. EPSTEIN,' M. TAKIZAWA,' B. STEIN,' I.A. KATZ,' 1.1. JOFFE,' D.F. ROMERO,' X.G. LIANG,2 M. LI,* H.Z. KE,2 W.S.S. JEE,3 T.W. JACOBS,' and J. BERLIN4

ABSTRACT

Cyclosporin A (CsA) is widely used in diabetic transplant patients and early type I diabetes mellitus. Diabetes produces a low-turnover osteopenia, and CsA conversely induces high-turnover osteopenia in rats. We investigated whether CsA would exacerbate diabetic osteopenia. Four groups of 10-week-old male Sprague- Dawley rats (n = ll/group) were studied: On day -6, groups A and C received saline and groups B and D received intravenous streptozotocin (55 mg/kg) to induce diabetes. From day 0, groups A and B received CsA vehicle and C and D received CsA (15 mg/kg) by daily gavage. Rats were bled on days -6, 0, 11, and 22 for serum bone gla protein (BGP), 1,25-(OH),D, PTH, blood ionized Ca, and blood glucose determinations. Double tetracycline labeling was performed on days 9 and 20 for bone histomorphometry. After sacrifice on day 22, histomorphometric analysis was performed. Serum BGP, 1,25-(OH),D, and PTH levels were significantly decreased in the diabetic alone (B) and diabetic plus CsA (D) groups and significantly increased in the CsA alone (group C). CsA alone (group C) induced cancellous bone loss by stimulated bone resorption. Cancellous bone loss in the diabetic alone rats (group B) was caused primarily by inhibited bone formation. No differences were found in cancellous bone mass, formation, or resorption parameters between diabetic alone (group B) and CsA-treated diabetic rats (group D). Neither CsA alone (group C) nor diabetic alone (group B) nor their combination affected cortical bone mass. CsA alone (group C) stimulated periosteal bone formation and endocortical bone resorption and inhibited endocortical formation, and diabetic alone (group B) inhibited both periosteal and endocortical bone formation. No parameters of tibia1 diaphyses in CsA-treated diabetic rats (group D) were different from diabetic alone. Thus the addition of CsA to the diabetic treated rats (group D) could not stimulate remodeling and appeared not to worsen significantly some of the alterations in bone formation and resorption. Possible explanations for this may be that CsA in vivo requires adequate levels of PTH, 1,25-(OH),D, insulin, and perhaps growth factors to stimulate remodeling. The use of CsA in type I diabetic patients or in organ transplant recipients who remain diabetic after transplantation may in the short term not aggravate existing osteopenia based on these findings.

INTRODUCTION significant osteopenia .(3,4) In addition, low-turnover osteopenia has been demonstrated in experimental diabetic animal mod-

EVERAL STUDIES have shown that type 1 (insulin-dependent) els."~" Our laboratory has shown decreased bone mass associ- S diabetes mellitus in humans is associated with reduced bone ated with significant reduction in serum bone gla protein (BGP), mass (osteopenia)."." Up to 50% of diabetic children have a marker of bone turnover and osteoblastic function, and serum

'Department of Endocrinology, Albert Einstein Medical Center. Philadelphia, Pennsylvania. 'Bone Biology Laboratory, Guangdong Medical College, Guangdong Province, People's Republic of China. 'Division of Radiobiology, University of Utah, Salt Lake City, Utah. 4Department of Biostatistics, University of Pennsylvania, Philadelphia, Pennsylvania.

557

558 EPSTEIN ET AL.

1,25-dihydroxyvitamin D [ 1,25-(OH),D] levels in experimental (streptozotocin-induced) diabetic rats."' Spontaneously diabetic rats (diabetic BB/W) with insulin-dependent diabetes also exhibit impaired bone turnover and osteocalcin prod~ction.'~.''

Cyclosporin A (CsA), a widely used immunosuppressive agent responsible for improving organ transplant survival in the past decade, has been administered to patients with a wide variety of immune-related diseases, such as rheumatoid arthri- tis, idiopathic uveitis, and psoriasis, as well as type I diabetes."' The rationale for its use in diabetes is that T cells play a central role"-l I ) ' in the (3 cell destruction of the pancreas and that CsA can modify or prevent this process if administered at the onset of the disease."2-17' This is confirmed in animal In addition, CsA is regarded as the drug of choice to prevent rejection in diabetic transplant patients (e.g., renal and pancreas). (')

The use of CsA is not without adverse side effects, however, such as nephrotoxicity and hepatotoxicity.") We previously reported that in vivo in the rat CsA produces severe osteopenia associated with a high-turnover state, with increased levels of serum BGP and serum 1,25-(OH),D and dramatic changes in bone histomorphometry with increased osteoclastic and osteoblastic activity.'2o'

Because of the use of CsA in diabetes and the results of our animal studies, we set out to determine whether CsA could exacerbate or modify the low-turnover osteopenia associated with type I diabetes. The clinical significance of this would have important ramification for diabetic patients receiving this immunosuppressive agent either to treat the disease or to prevent transplant organ rejection in recipients with diabetes.

MATERIALS AND METHODS

Animals

A group of 44 male Sprague-Dawley rats aged 10 weeks, weighing approximately 350 g, were obtained from Charles River Laboratories (Wilmington, MA). All rats were housed under similar conditions and maintained on a diet of Agway Prolab RMH 3000 (0.75% calcium, 0.85% phosphorus, 1045 IU/kg of vitamin D,, 22.5% protein, 5.5% fat, and 52% carbohydrate) and tap water ad libitum.

Drugs

CsA solution (Sandoz, Inc., East Hanover, NJ), in a concentration of 100 mg/ml of 12.5% alcohol by volume and olive oil, was diluted in alcohol-olive oil vehicle to obtain a final concen- trationof 15 mglml. Streptozotocin (Sigma, St. Louis, MO) was dissolved in saline at a concentration of 55 mg/ml immediately before use.

Experimental protocol

Rats were randomly divided into four groups and were treated according to the following protocol: on day -6, groups A (n = 1 I ) and C (n = 11) received saline (100 pl / IOO g body weight) and groups B (n = 11) and D (n = 1 1 ) received streptozotocin (55 mg/kg/body weight) by a single saphenous vein injection. On day 0, all animals were bled from the tail vein for measure-

ment of blood glucose. Animals in groups B and D with a blood glucose less than 16.8 mM were excluded from the study. From day 0 through day 22, groups A and B received CsA vehicle (100 p1/100 g body weight) by daily gavage and groups C and D received CsA (15 mg/kg body weight) by daily gavage.

All rats received tetracycline hydrochloride (Achromycin; Lederle Laboratories, Pearl River, NY), 15 mg/kg body weight by subcutaneous injection on days 9 and 20, for determination of histomorphometric parameters.

Experimental procedures were reviewed and approved by an institutional animal care committee in accordance with current National Institutes of Health policy.

Blood collection and analysis

Rats were weighed and bled on days -6, 0, 11, and 22 after 10 mg/100 g body weight of ketamine hydrochloride (Ketaset; Aveco Co., Inc., Fort Dodge, 1A) and 0.1 mg/ 100 g body weight of acepromazine maleate (Techamerica Group Inc., Elwood, KS) administered intramuscularly. On day 22, tail vein blood was collected in heparinized capillary tubes for immediate blood ionized calcium measurement by an ICAl ionized calcium analyzer (Radiometer Co., Copenhagen, Denmark). Tail vein blood was also used to measure blood glucose with an Accuchek blood glucose-measuring device (Boehringer Mannheim Diag- nostics, Indianapolis, IN). All other blood samples were obtained via orbital sinus venous puncture with nonheparinized pasteur pipettes, except when rats were sacrificed by cardiac puncture on day 22. Blood samples were centrifuged and the sera stopped at -70°C until assayed.

Serum bone gla protein was measured by radioimmunoassay (RIA) using a modification of a previously described assay."" Antibody to rat BGP was raised in rabbits immunized by multiple-site intradermal injections of purified rat BGP. The antibody was used in a final dilution of 1:25,OOO. Purified rat BGP was used for the preparation of standards and '*'I-labeled tracer. After a 24 h equilibrium assay, antibody-BGP complexes were separated from free 1Z51-labeled BGP with goat antirabbit gammaglobulin (Calbiochem, Lalolla, CA). The lower limit of detection for the assay is 0.5 ng/ml, and the intra- and interassay coefficients of variation are 5.9 and 9.8%, respectively.

Serum immunoreactive parathyroid hormone (PTH) was measured by RIA using a commercially available kit (N- terminal PTH; Nichols Institute, San Juan Capistrano, CA) with an antiserum to human PTH-( 1-34) that cross-reacts with rat PTH-(1-34). The validity of this assay has been confirmed in our laboratory by showing low values for serum immunoreactive PTH in parathyroidectomized rats and elevated values in EDTA- induced hypocalcemic rats. The characteristics of this assay have been published.'22' Intra- and interassay coefficients of variation are 8.2 and 10.4%, respectively.

1,25-Dihydroxyvitamin D was assayed using a commercially available radioreceptor assay kit (Incstar, Stillwater, MN) specific for both 1,25-(OH),D, and 1,25-(OH),D,. Intra- and interassay coefficients of variation are 3.5 and 8.5%, respectively.

Histological techniques

Following sacrifice, the right tibia was removed from each rat, dissected free of soft tissue, and fixed in 70% ethanol. The

CYCLOSPORIN AND BONE METABOLISM IN DIABETIC RATS 559

proximal tibial metaphysis and tibial shaft (proximal to the tibiofibular junction) of each tibia were stained in Villanueva bone stain (Polysciences, Inc., Warrington Pa) for 5 days. Specimens were dehydrated by sequential changes in ascending concentrations of ethanol and acetone and then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester NY). Frontal sections (230 pm thick) of the proximal tibia and cross sections of tibial shaft were cut with a precision bone saw. Two sections from each tibia were ground to 100 p m thickness and microradiographed on Kodak spectroscopic plates (649; Eastman Kodak, Rochester NY).‘”’ The 100 pm thick sections were further ground to a thickness of 20 and 30 pin for static and dynamic histomorphometric measurements.‘27-25)

A digitizing image analysis system was used to determine selected static and dynamic proximal tibial metaphyseal cancellous bone and tibial shaft cortical bone histomorphometric indices. The system consists of an epifluorescent microscope, a digitizing pad (Summagraphic, Fairfield, CT) coupled to an Apple Maclntosh SE computer, and the morphology program Stereology (KSS Computer Systems, Magna, UT). To evaluate the changes in cancellous bone, the total tissue area, trabecular area and perimeter. eroded perimeter (active and inactive resorption surfaces), osteoid perimeter, single- and double-labeled perimeter, and interlabeling distance of double fluorescent labels (at the trabecular bone surface and the growth plate- metaphyseal junction region) were measured in the region (between 1 and 4 mm distal to the growth plate-metaphyseal

The percentage trabecular area, number, and width, percentage eroded, osteoid and labeling perimeters, mineral apposition rate, bone formation rates, longitudinal growth rate, and the ratio of percentage labeling perimeter and ended perimeter were calculated.‘23-2x’ For the cortical bone evaluation, histomorphometric measurements were performed on 30 p m cross sections of tibial diaphyses. Total tissue area, percentage marrow and cortical bone areas, periosteal and endocortical labeling perimeter, mineral apposition rate, bone formation rate, and endocortical eroded perimeter were determined. The measured and derived parameters are listed and defined in Table 1.

Statisticul methods

Statistics were calculated using the statistics packages SPSSI PC+ (SPSS, Inc., Chicago, 1L) and SAS (SAS Institute, Inc., Cary , NC). Repeated-measures analysis was performed with multivariate analysis of variance. Analysis of the bone histomorphometry was performed with a 2 x 2 factorial analysis of variance (ANOVA), including a priori pairwise contrasts. Both models included interactions between CsA and diabetes. The test of the CsA-diabetes interaction assesses whether the magni- tude of any alterations in the biochemical or bone histomorphometric parameters induced by CsA depends on the presence or absence of diabetes. The pairwise contrasts test differences in mean bone parameters between specific groups of rats in the context of the 2 x 2 factorial experiment. A p value < 0.05 was considered a significant difference. All values are reported as the mean * standard error of the mean (SEM) in both tables and figures. Even when the 2 X 2 interaction term for the interaction of CsA and diabetes is not significant, the differences between groups are reflected accurately by the pairwise contrasts.

RESULTS

Bloodglucose: On day -6 ofthe experiment, all groups had similar blood glucose levels (Fig. I ) . From day 0 and for the duration of the experiment, both diabetic groups (B and D) had blood glucose levels persistently above 23.5 mM, but we observed that the CsA alone (group C) had progressively increasing blood glucose levels, and on day 22 the final level was above 19.6 mM.

Blood ionized calcium: At the beginning of the experiment all groups (A-D) had similar blood ionized calcium levels (Fig. 2 ) . On day 2 2 , the CsA alone rats (group C) and the diabetic and diabetic plus CsA groups (B and D) had significantly lower ionized calcium levels than the control group (A). There was no difference in ionized calcium levels between groups B and D.

Body weight: The control group (A) and cyclosporin alone group (C) increased body weight progressively, but the diabetic alone group (B) and diabetic plus CsA group (D) lost significant weight compared with the control and CsA alone groups of rats (Fig. 3 ) . However, the weight loss was similar in both the diabetic alone (B) and diabetic plus CsA group (D) at the end of the study (Fig. 3 ) .

Serum BGP: All groups had similar serum BGP values at day -6, but already at day 0 the diabetic alone group (B) and the diabetic plus CsA treatment group (D) had significantly decreased BGP values compared with the control group (A) as well as the CsA alone group (C) (Fig. 4). This difference persists throughout the study periods. In the CsA alone treated group (C), BGP continued to increase from day 0 through day 2 2 .

Serum PTH: At day 0 the diabetic alone group (B) and diabetic plus CsA group (D) had elevated serum PTH values that presumably reflected the decreased calcium values (Fig. 5). When the diabetes became established, PTH levels significantly decreased compared with the control group (A) and the CsA alone group (C).

Serum f ,ZS-(OH),D: The serum I ,25-(OH),D values at day 0 and 22 were significantly decreased in both groups of diabetic animals (groups B and D) compared with control (A) and CsA alone groups (C) (Fig. 6). In the CsA alone group (C). serum 1,25-(OH),D (as found in our previous studies) was significantly elevated on day 2 2 .

Bone histomorphometr?,

In proximal tibial metaphyseal cancellous bone (Table 2 ) , CsA alone decreased trabecular bone area (p < 0.01). trabecular number (p < 0.05). and ratio of labeled to eroded perimeter (p < 0.01) and modestly increased eroded (resorption) perimeter (p < 0.05) compared with age-related controls. In addition, we found that CsA stimulated an increase in longitudinal growth rate (p < 0.01). The diabetic alone rats showed significant decreases in trabecular bone area (p < 0.05), trabecular thickness (p < O.Ol) , percentage osteoid perimeter (p < 0.001), mineral apposition rate (p < 0.001). labeling perimeter (p < 0.001). bone and tissue-referent bone formation rate (p < 0.001), and longitudinal growth rate (p < O.OOl), as well as a significant increase in percentage eroded perimeter (p < 0.05), compared with age-related controls. In 3 of 9 diabetic rats and 3 of 10 CsA-treated diabetic rats the proximal tibial cancellous bone

560 EPSTEIN ET AL.

TABLE 1. HISTOMORPHOMETRIC MEASUREMENTS AND CALCULATIONS

Parameters Abbreviations Formulas Units

I. Cancellous bone parameters A. Measurement

Total tissue area Trabecular area Trabecular perimeter Single-labeled perimeter Double-labeled perimeter Interlabeled width Interlabeled width in growth plate Eroded perimeter Osteoid perimeter

% trabecular area Trabecular thickness Trabecular number Trabecular separation % labeled perimeter % eroded perimeter % osteoid perimeter Mineral apposition rate Bone formation rates (bone area

Bone formation rates (tissue area

Longitudinal growth rate Ratio of labeled to eroded

B. Calculations

referent)

referent)

perimeter 11. Cortical bone parameters

General Total tissue area Marrow area Periosteal perimeter Endosteal perimeter Cortical area % Cortical area % Marrow area

Periosteal surface Single-labeled perimeter Double-labeled perimeter Interlabeled width Osteoid perimeter % labeled perimeter % osteoid perimeter Mineral apposition rate Bone formation rate

Single-labeled perimeter Double-labeled perimeter Interlabeled width Eroded perimeter % labeled perimeter % eroded perimeter Mineral apposition rate Bone formation rate (surface)

Endocortical surface

T.Ar Tb.Ar Tb.Pm sL.Pm dL.Pm Ir.L.Wi Ir . L. Wi-G Er . Pm O.Pm

% Tb.Ar Tb.Th Tb.N Tb.Sp %L.Pm %Er.Pm %O . Pm MAR BFR/BV

BFR/TV

L.G.R. L. Pm/Er. Pm

T.Ar Ma.Ar P-Pm E-Pm Ct.Ar % Ct.Ar % Ma.Ar

sL.Pm dL.Pm Ir.L.Wi O.Pm %L.Pm % O.Pm MAR BFR/BS

sL.Pm dL . Pm Ir.L.Wi Er.Pm %L.Pm %Er.Pm MAR BFR/BS

Tb.Ar/T.Ar X 100 (2000/1.199) X Tb.Ar/Tb.Pm 1.199/2 X Tb.Pm/T.Ar (2000/1.199 X (T.Ar-Tb.Ar)/Tb.Pm L.Pm/Tb.Pm X 100 Er.Pm/Tb./Pm X 100 O.Pm/Tb.Pm x 100 1r.L. Wihnterval (dL.Pm + sL.Pd2) X MAR/Tb.Ar X 365 X 100 (dL.Pm+sL.Pm/2) x MAR/T.Ar x 365 x 100

mm2 mm2 mm mm mm Pm Pm mm mm

% (*m #/mm (*m % % %

%/year

%/year

PddaY

Ir.L.Wi-G/interval PddaY Percent labeled perimeter/percent eroded Ratio perimeter

h

h

b

b

- - -

-

T. Ar-Ma. Ar (T.Ar.-Ma.Ar)/T.Ar. X 100 Ma.Ar./T.Ar. X 100

mm2 mm2 mm mm mm2 % %

h

h

h

h

- mm mm Pm mm

(dL.Pm + sL.Pm/2)/P-Pm X 100 % O.Pm/P-Pm X 100 % 1r.L. Wi/interval PddaY (dL.Pm + sL.Pd2) X MAWP-Pm X 100

- - -

pm/day x 100

h

h

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h

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-

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“Definitions: T.Ar, total tissue; Tb.Ar, total cancellous bone area within T.Ar; Tb.Pm, perimeter of Tb.Ar; sL.Pm, length of trabecular surface with tetracycline; dL.Pm, length of trabecular surface labeled with tetracycline and calcein; 1r.L. Wi, distance between tetracycline and calcein labels; 1r.L. Wi-G, distance between tetracycline and calcein labels (growth plate metaphyseal junction regions); Er.Pm, length of trabecular surface with Howship’s lacunae; O.Pm, length of trabecular surface with osteoid.

’Measured parameters.

CYCLOSPORIN AND BONE METABOLISM IN DIABETIC RATS 56 1

0-0 - Control 0-0 - CsA A-A = Diobetic

= p ( 0.05 vs Control I = p ( 0.05 vs CsA

I A-A = Diobetic + CsA

E v 22.4 VI 0 V

16.8 d n

m

0 2 11.2

5.6 i - -6 0 1 1 22

DAYS

FIG. 1. Blood glucose levels (mM) were elevated at days 0, 1 I , and 22 in the diabetic and diabetic plus CsA groups compared with control and CsA groups. The groups receiving CsA alone also had significantly elevated blood glucose levels at days I I and 22 compared with control.

failed to label with tetracycline. This failure to take up injected tetracycline was previously reported by Shires et al.'zy' and Goodman and Hori.'"" CsA did not alter diabetes-induced changes in static or dynamic parameters of cancellous bone. All parameters listed in Table 2 were not significantly different between diabetic alone (group B) and the diabetic rats treated with CsA (group D).

In tibial shaft cortical bone (Table 3), CsA treatment (group C) did not influence total tissue area, percentage cortical bone area, or percentage marrow area; however, the dynamic data showed that CsA increased periosteal bone formation rate @ < 0.05) and labeling perimeter (p < 0.05) but inhibited endocortical bone formation rate @ < 0.01) and endocortical mineral apposition rate 0, <I 0.01) and stimulated endocortical resorption (increased eroded surface; p < 0.001). In diabetic rats (group B), total tissue area (p < 0.05) and periosteal0, < 0.001) and endocortical bone formation 0, < 0.001 ) were significantly lower, but endocortical eroded perimeter was not different from that in age-related controls. The frequency of double label in the periosteal bone of tibial shafts was I I of 1 I in normal controls and CsA-treated rats, 5 of 8 in diabetic rats, and 8 of 12 in

1.360 , - p ( 0.05 vs Control

0 w E H 1.300 0

1.270

Control CSA Diabetic Diobetic + CSA

FIG. 2. Blood ionized calcium (Ca) was significantly decreased on day 22 in the CsA-treated, diabetic, and diabetic plus CsA groups compared with control. There was n o difference between the diabetic and diabetic plus CsA groups.

500

450 - 8 400 0 t- v

350 0 0 m

0-0 CONTROL = P ( 0.05 VS. CONTROL 0-0 = CSA

A-A = DIABETIC T

A-A = DIABETIC + CSA

I . - P ( 0.05 VS. CSA

/?

250 0,

-6 0 1 1 22

DAYS

300 t FIG. 3. There was a significant reduction in body weight in the diabetic and diabetes plus CsA group at days 0, I I , and 22 compared with control and CsA-treated rats.

CsA-treated diabetic rats. Endocortical double labels were observed in 10 of I I normal control rats, 8 of 1 I CsA-treated rats, 1 of 8 diabetic rats, and I of 12 CsA-treated diabetic rats. All parameters listed in Table 3 were not significantly different between diabetic alone (group B) and the combination of diabetes plus CsA (group D).

DISCUSSION

This study demonstrates that experimental diabetes in rats produces profound effects on bone mineral metabolism. Diabe- tes caused a significant alteration in calcium and calciotropic hormones, with decreases in serum PTH and 1,25-(OH),D. These findings confirm what we and others showed previously, except that in our original study we did not demonstrate a decrease in FTH,'" but we think this may either be a transient effect or due to the different calcium content of the diet and younger age of the rats, as well as the duration of the experiment

200

- 150

> 4 100 v

m I 3 u:

50

0-0- Control 0-0 = CSA A-A= Diobetic

= p ( 0.05 vs Control I . - p ( 0.05 vs CsA

A-A= Diabetic + CSA

0 -6 0 1 1 22

DAYS

FIG. 4. There was a significant decrease in serum bone gla protein (BGP) in the diabetic and diabetic plus CsA groups at days 0, 1 I , and 22 compared with control and the CsA group. The CsA group had significantly increased levels at day 1 I and 22 compared with control rats.

562 EPSTEIN ET AL.

0-0 = Control 0-0 = CSA A-A = Diabetic A-A = Diabetic + CSA

= p ( 0.05 vs Control 1, = p ( 0.05 vs CSA

50 I -6 0 22

DAYS

FIG. 5. Serum PTH was significantly increased at day 0 in the diabetes and diabetes plus CsA groups and decreased at day 22 in the diabetes plus CsA group and diabetic group compared with the control group and the CsA group.

(49 as opposed to 22 days) in the current study. The reason for the decreased PTH and 1,25-(OH),D levels in diabetes is not clear, although insulin and PTH have been shown to influence the la-hydroxylase system in the kidney.'"'.") The possibility of severe hyperglycemia affecting FTH and 1,25-(OH),D rne- tabolism cannot be excluded, although this is unlikely because hyperglycemia occurred in the CsA-treated rats without streptozotocin and serum PTH and 1,25-(OH),D levels were normal or increased, respectively, not decreased. The decrease in these calciotropic hormones almost certainly played a major influence in the growth, modeling, and remodeling events in the bone of the diabetic rats seen in our study over the 22 day period, although there was an increase in eroded trabecular surface that is not consistent with the lowered PTH and 1,25-(OH),D levels.

We measured total circulating serum 1,25-(OH),D in our study, but Nyombaet al. showed that, in fact, free 1,25-(OH),D values are normal in diabetic rats and that the decrease may be due to a decrease in D binding proteins.(32' However, there may

120

h z n n 2 60 I 0 In

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0

FIG. 6.

0-0 = Control 0-0 = CSA A-A = Diabetic A-A = Diabetic + CsA

= p ( 0.05 vs Control L = p ( 0.05 vs C d

.

-6 0 DAYS

22

Serum 1 ,25-(OH)2D values were significantly decreased at day 0 and day 22 inihe diabetic and diabetes plus CsA groups compared with control and CsA groups. In the CsA group serum 1,25-(OH),D was increased significantly at day 22 compared with the control group.

be resistance of the insulinopenic bone to normal circulating levels, and the decreased serum BGP reflects this resistance with reduced osteoblastic activity and decreased rates of formation. The administration of CsA could not stimulate the la-hydroxylase system and the production of 1,25-(OH),D, despite increasing it in the rats treated with CsA alone. CsA increases the production rate of 1,25-(OH),D without altering the metabolic clearance rate (MCR) in the rat,'") and the lack of response in the CsA-treated diabetic animal may be because insulin, insulin- like growth factor I (IGF-I), and PTH are required in addition for enhanced synthesis of I ,25-(OH),D or that perhaps the MCR of 1,25-(OH),D is increased in the CsA-treated diabetic rat. The latter is unlikely because the MCR has been reported to be normal, and free 1,25-(OH),D values are either normal or increased in the diabetic rat.'32'

Similarly, the decrease in serum BGP reflects the diabetic state with diminished osteoblastic function. This reduced activity again may reflect the lack of insulin, local growth factors, such as IGF-I, and/or a resistance to the action of normal free levels of I ,25-(OH),D. CsA, which is a very potent stimulus of BGP,'*'' increased levels in the CsA only (group C) treated rats but was unable to elevate levels in the presence of diabetes. Hyperglycemia, as mentioned earlier, did not influence the BGP levels; the CsA only treated rats (group C) had excess circulating levels of glucose as well as BGP. BGP is an indicator of osteoblast activity and bone remodeling, and we previously found it to be a good reflection of the histomorphometric finding^.'^' This was again demonstrated in this study, with indices of growth, modeling, and remodeling increased in the CsA alone treated rats but decreased in the diabetic rats with decreased serum BGP levels.

The bone histomorphometric findings in the diabetic animals were somewhat similar to our previous and other findings: a bone loss and massive decrease in bone formation."-2y-3"0'These studies differ somewhat in resorption indices. Our previous and the Shires et al.'29' findings reported a decrease in osteoclast number and the Verhaeghe"' and Goodman and Hori'"' studies reported no change in osteoclast numbers and endosteal resorption rate, but the current study demonstrated some increase in eroded cancellous bone surface. The increased eroded surface in diabetic rats can best be explained by a defective coupling of bone formation to resorption in which bone resorption is fol- lowed by a prolonged period of bone formation, and this has been reported by Takahashi and Frost in diabetic patient^."^' It would also explain why the eroded trabecular surface in the current study was increased but the number of osteoclasts was decreased in previous

In cortical bone, the responses in bone formation were similar to those in cancellous bone, with reduced periosteal and endocortical bone formation. The net result in both cancellous and cortical bone was osteopenia. The diabetic rats lost weight, but weight loss as discussed in detail in our previous studies, as well as those by Verhaeghe et al.'6,7' and Shires et a1.,'35' does not account for this sustained decrease in remodeling. This type of remodeling contrasts with that found with CsA, in which excessive turnover is seen on the periosteal surface, with labeled perimeter (LPM%) and bone formation rate (BFR) increased and cancellous bone excessively resorbed. The net result in this CsA model is osteopenia with loss of cancellous bone volume. These findings overall reflect our previous results.'20' It is thus swpns-

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2.40

o.O

o01

199.

57

9.41

23

3.50

0. I1

06

42.0

2 4.

20

32.8

4

O.oO

01

0.68

0.

12

0.27

o.oO

01

3 I .6

2 6.

I3

8.79

o.O

o01

1.71

0.

34

17.5

9

0.22

-1

0.

I7

-3

0.95

0.

95

-2

9 7.

49

2.41

-3

2h

13h

0.13

3

14.6

2 17

h 2.

00

- 22

0.

06

-61'

I .

96

- 72

' 3.

10

929"

%

ver

sus

cont

rol

Dia

betic

s SE

M

% v

ersu

s co

ntro

l %

ver

sus

CsA

D

iabe

tics

and

CsA

SE

M

% v

ersu

s co

ntro

l %

ver

sus

CsA

%

ver

sus

Dia

betic

s

8 5.

58

0. I3

- lo

h -

4.46

0.

17

1 2h

79.7

8 20

.50

1.63

I .

06

-1

7

0.01

56

.20

0.00

3.

22

- 99

d - 34

d

- lo

od

-42d

0.78

0.

12

~ 66

"

43.7

2 6.

10

- 78

"

32.3

2 3.

35

- 23

0.07

0.

05

- 90

*

3.63

2.

76

- 89

*

3.48

1.

11

104

-lob

-9

1 -2

- 67

" -8

1d

-2

-73

- 59

-so

d 12

5.

84

4.58

78

.51

21.4

9 0.

03

64.6

0 0.

54

34.5

2 41

.82

0.05

2.

80

4.78

0.09

-6

0.

10

-9

1.25

I .

25

-3

12

0.01

3.

88

- 99

'J - 25

'j

- lo

od

- 33

'' 18

4 15

0.06

- 77

d 4.

32

-83d

4.

29

0 0.

04

-93d

2.

02

-91'

J I .

34

I80

-6 5

-6 3

-1

3 -2

5

- 77

'' -3

1 -8

9

-21

27

29

-81

- 29

- 68

- 23

-7

3d

37

an

= n

umbe

r of

'p

< 0.

05.

'p <

0.0

1.

'Jp <

0.0

01.

anim

als.

TA

BL

E 2. S

TA

TIC

AN

D D

YN

AM

IC

HIST

OMOR

PHOM

ETRY

O

F PR

OX

IMA

L T

IBIA

L

MET

APH

YSEA

L CAN

CE

LL

OU

S BO

NE (

20 p

M S

EC

TIO

NS)

"

% L

abel

ing

Min

eral

B

one

Bon

e pe

rim

eter

1

area

th

ickn

ess

num

ber

sepa

ratio

n pe

rim

eter

pe

rim

eter

ra

te

peri

met

er

rate

(B

V)

rate

(TV)

grow

th r

ate

peri

met

er

Trab

ecul

ar

Trab

ecul

ar

Trab

ecul

ar

Trab

ecul

ar

Ero

ded

Ost

eoid

ap

posi

tion

Labe

ling

form

atio

n fo

rmat

ion

Long

itudi

nal

erod

ed

Gro

up

(%)

(pm

) (#

lmm

)-'

(pm)

(%)

(%)

(pm

lday

) (%

) (%

/yea

r)

(%/y

ear)

(p

rnld

ay)

(rat

io)

AN

OV

A

0.04

46

0.00

1 0.

0680

0.

1638

0.

0380

C

ontr

ol

11

9.86

50

.05

1.91

53

5.78

7.

32

SEM

1.

39

1.97

0.

20

64.8

4 0.

96

CsA

12

5.

54

48.2

9 1.

10

1149

.76

10.5

4 SE

M

I .02

2.

05

0. I7

18

1. I9

0.

68

% v

ersu

s -4

4'

-4

-42'

1 I

5 44

' co

ntro

l D

iabe

tics

9 6.

51

40.1

4 1.

70

1077

.88

10.6

2 SE

M

1.18

3.

60

0.29

43

0.05

0.

95

% v

ersu

s - 34

' - 20

b -1

1 10

1 45

'

% v

ersu

s CsA

18

- 17

' 55

-6

1

Dia

betic

s an

d 10

6.

32

34.8

8 I .

73

804.

62

10.7

3

SEM

1.

05

I .45

0.

26

21 1

.39

0.99

%

ver

sus

- 36

' - 3

0d

- 10

50

47

'

% v

ersu

s C

sA

14

- 2g

d 57

- 30

2

% v

ersu

s -3

- I3

I

- 25

1

cont

rol

CsA

cont

rol

diab

etic

s

an =

num

ber o

f ani

mal

s.

'p <

0.05

. 'p

< 0.

01.

dp <

0.00

1.

o.O

o01

o.O

o01

o.O

oo1

o.O

oo1

o.O

oo1

o.O

o01

o.O

oo1

13.9

4 1.

71

25.9

4 55

2.00

49

.95

9.50

4.

43

1 SO

0.

12

1.93

57

.84

6. I9

0.

58

0.78

13

.07

1.76

27

.95

625.

57

33.8

2 13

.70

2.75

I .

96

0.11

I .

73

54.2

6 6.

22

0.28

0.

20

-I

3 8

13

-32'

44

h -3

8'

4.23

0.

29

9.73

44

.01

2.35

4.

00

0.92

I .

96

0.04

1.

58

8.26

0.

43

1.05

0.

11

- 70

" -8

3"

- 62

" - 92

d -9

9

-5@

- 79

"

- 6g

d - 83

" - 65

d - 93

d -9

3'

-71'

-6

7'

2.79

0.27

11

.22

59.7

7 3.

05

5.09

I .

08

0.55

0.

03

1.62

16

.46

0.64

I .

72

0. I3

-8

0"

- 84

d - 57

" - 89

" - 94

" - 46

* - 76

*

- 79

d -8

5d

- 60

" - 90

d -9

1d

-63d

-6

1'

- 34

-9

15

36

30

27

17

CYCLOSPORIN AND BONE METABOLISM IN DIABETIC RATS 565

ing in view of the profound effect of CsA on cancellous bone that in the diabetic rat there was no greater evidence of resorption and formation and further bone loss (Tables 2 and 3). The possible explanation may be that CsA requires insulin as well as 1,25- (OH),D and PTH to stimulate remodeling. The hyperglycemia may also play a role, that is, a diabetes-like effect, but this is unlikely because the CsA group, despite hyperglycemia, had increased remodeling. This points to insulin deficiency as a major factor, which in turn in streptozotocin rats is aggravated by C S A . ' ~ ~ ' We did not measure insulin levels in our animals, so this is unproven. Another explanation may be that growth factors and the cytokine lymphokine pathway may be abnormal in the insulinopenic state and the action of local bone-resorbing and bone-forming factors, such as interleukins 1 and 6, tumor necrosis factor, and transforming growth factor p may be compromised. We did not assay these factors, so that this explanation remains speculative. In addition, the phannacoki- netics of CsA (i.e., biologic availability) may also be altered in diabetes, which could have affected the bone remodeling. We did not measure circulating levels of cyclosporin, but even normal or increased levels may not definitely exclude an impaired biologic action of CsA in diabetic rats. A further issue that may also have influenced our biochemical and bone histomorphometry is the elevation of glucocorticoids that can occur in streptozotocin-treated rats'37) and may possibly have suppressed osteocalcin and bone remodeling.'")

If one extends these results to the clinical situation. it suggests that in insulin-dependent diabetic patients, in whom osteopenia is evident, or in organ transplant patients who remain diabetic posttransplantation the addition of CsA therapy to these patients may not contribute to further bone loss. This was a short-term study, however, and the dynamics of bone turnover may well alter over a prolonged period of time. In any event, these results suggest that certain patients may well warrant future observation and scrutiny with regard to bone mass.

ACKNOWLEDGMENTS

We thank Ms. Mary Wingfield for her secretarial skills. Funds were made available by the U.S. National Aeronautics and Space Administration (NAG 2-435); U.S. Department of Energy and the University of Utah (DE-FG02-89ER 60764); the National Institutes of Health (AR-38346); and the U.S. Depart- ment of Energy Contract (DE-AC02-76EV-00119).

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Address reprint requests to: Sol Epstein, M.D.

Division of Endocrinology and Metabolism Albert Einstein Medical Center

5401 Old York Road Philadelphia, PA 19141

Received in original form April 7, 1993; in revised form July 26, 1993; accepted July 27, 1993.

Effect of cyclosporin A on bone mineral metabolism in experimental diabetes mellitus in the rat

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