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Calcif Tissue Int (1985) 37:605-607

a l c i f i e d T i s s u e

I n t e rn a t i o n a l

9 1985 by Springer-Verlag

o n e D i s o r d e r s i n S p o n t a n e o u s l y H y p e r t e n s i v e R a t

Y o s h i h ir o I z a w a K i y o s h i S a g a r a T a k a sh i K a d o t a a n d T o k u t a r o M a k i t a

Teijin Institute for Bio-medical Research, 4-3-2 Asahigaoka, Hino-city, Tokyo 191, Japan

S u m m a r y . T h e b o n e s o f a d u lt 2 6 w e e k s o l d ) s p o n -

t a n e o u s l y h y p e r t e n s i v e ra ts S H R ) w e r e e x a m i n e d

c h e m i c a l l y a n d h i s t o l o g i c a l l y b y c o m p a r i n g t h e m

w i t h t h o s e o f th e c o r r e s p o n d i n g n o r m o t e n s i v e

W i s ta r -K y o to W K Y ) ra t s . T h e mea n co r t i ca l th i ck -

n e s s , t h e m e a n a s h w e i g h t p e r u n it b o n e v o l u m e ,

a n d t h e a s h a s p e r c e n t a g e o f d r y w e i g h t o f f e m u r

w e r e s i gn i f ic a n t ly l o w e r i n S H R t h an i n W K Y . B e -

s i d es , th e p ercen t co r t i ca l a rea mea s u red o n t i b i a l

c r o s s - s e c t i o n w a s a l s o r e d u c e d i n S H R c o m p a r e d

w i th W K Y . T h es e f i n d i n g s s t ro n g l y s u g g es t th a t th e

d e v e l o p m e n t o f o s t e o p o r o t i c b o n e d i s o r d e r s e x i s t s

i n a d u l t s p o n ta n eo u s l y h y p er ten s i v e ra t s .

K e y w o r ds : S H R - - W K Y - - O s t e o p o r o si s - - B o n e .

Introduct ion

R ecen t s tu d i e s h a v e d emo n s tra ted th a t th e meta b -

o l i sm o f c a l c iu m i s a b n o r m a l in h y p e r t e n s i o n . H o w -

ev er , l it t le a t t en t io n h a s b ee n fo cu s ed o n a s s o c i a t i o n

b e t w e e n h y p e r t e n s i o n a n d b o n e d i s o r d e r s . I n t h e

p r e s e n t s t ud y , t h e r e f o r e , w e e x a m i n e d b o n e t i s su e s

b i o c h e m i c a l l y a n d h i s t o l o g ic a l l y i n n o r m o t e n s i v e

a n d s p o n ta n eo u s l y h y p er ten s i v e ra t s a t a n a g e a f t er

t h e d e v e l o p m e n t o f h y p e r t e n s i v e b l o o d p r e s s u r e.

T h e p r e s e n t o b s e r v a t i o n s p r o v i d e e v i d e n c e t h at o s -

t e o p o r o s i s i s d e v e l o p e d i n s p o n t a n e o u s l y h y p e r t e n -

s ive rats .

M a ter i a l s a n d M eth o d s

Ten male spontaneously hypertensive (SHR) and normotensive

Wistar-Kyoto (WKY) rats were obtained from Nippon Charles-

River Co. at 20 weeks of age. Average weight of rats was 376 g

in WKY and 356 g in SHR. Animals were housed individually

in cages and weighed weekly. They were fed ad libitum with a

standard laboratory diet (Nippon Crea Inc.) containing 1% cal-

cium and l% phosphorus with 2,000 units of vitamin D per kg

end reprint requests to Y. Izawa at the above address.

throughout the experiment. Systolic blood pressure (SBP) was

measured in each animal at 26 weeks of age before they were

sacrificed. Pressure was measured on the tail of unanesthetized

rats wit h Rat Tail Manometer-Tachomet er System (KN-210, Nat-

sume, Japan). Urinary calcium (Ca) and inorganic phosphorus

(Pi) excretions were measured for 24 h at 26 weeks. Then, blood

was drawn from the carotid artery under pentobarbital anes-

thesia. Serum Ca and Pi concentrations were determined. Ca

and Pi wer e analyzed using an autoanalyzer(Hitachi 706D).

The right and left femurs were removed and dissected free of

soft tissue, and the weight and length of the left were recorded.

The volume of the bone was determined by using plethysmo-

meter (KN-357, Natsume). Roentg enograms were taken with the

use of a Softe x (CSM), and from these , cortical thickness of the

femur was measured at the middle of the whole bone and ex-

pressed as % of the total width of the bone [1]. They were sub-

sequently dried in an oven at 110~ for 5 days for the measure-

ments of dry weight. Dried bones were ashed in porcelain cru-

cibles at 900~ for 5 h to obtain the ash weight. The right femurs

were used for the measurement of the cortical bone alone. The

bone marrows were washed away by water flush and a portion

of cortical bone from each rat was measured for the dry weight

and ash conte nt according to the same method described above.

The right tibias were fixed in 70% ethanol and embedded

without decalcification in polyest er resin (Ligorac Nissin EM

Inc.), and three serial cross-sect ions (50-60 ~xm) per one spec-

imen were cut at the point of one-third from the proximal end

of the bone. Then the sections were examined histomorpholog-

ically with contact microradiography (CMR). The tibia was

morphologically measured with an image analyzer [2].

Studen t's t-test was used to evaluate the significances between

S R an d W K Y groups.

Resul ts and Discuss ion

R e cen t s tu d i e s h a v e d emo n s tra ted th a t s eru m io n -

i zed C a co n ce n tra t i o n s w ere red u ced [3 ], s eru m i m-

mu n o rea c t i v e p a ra th y ro id h o rm o n e P T H ) l ev e l s i n -

crea s ed [4 ], a n d i n te s t in a l C a a b s o rp t i o n red u ced i n

t h e s p o n t a n e o u s ly h y p e r t e n s i v e r a ts S H R ) a s c o m -

p a r e d w i t h t h o s e i n t h e n o r m o t e n s i v e W i s t a r - K y o t o

ra t s W K Y ) [5 1 . S ch ed l e t a l . [5 ] h a v e a l s o recen t l y

rep o r ted th a t th e p o s s i b i l i ty o f a b n o rma l meta b o -

l i s m o f v ita mi n D m i g h t ex i s t in S H R . T h es e f i n d -

i n g s s t r o n g l y s u g g e s t p o s s i b l e a b n o r m a l i t i e s o f

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Table 1. Body weight, systolic blood pressure (SBP), and serum

concentration and urinary excretion of calcium and inorganic

phosphorus

WKY SHR

Numbe r of rats l0 10

Bod y wei ght (g) 408 14 386 24 ~

SBP (m mH g) 160 5 236 +_ 10 c

Ser um Ca (mg/100ml) 9.44 0.05 9.23 0.09

Se rum Pi (mg/100ml) 5.26 ~ 0.11 4.22 0.30 b

Urin ary Ca (rag/day) 0.78 0.04 0.54 0.03 ~

Ur inary Pi (rag /day ) 19.4 0.9 21.1 1.1

Values are mean s _+ SE. WKY, norm oten sive Wista r-Kyo to rat;

SHR, spontaneously hypertensive rat

a p < 0.05, bp < 0.01, cp < 0.00 1--sig nifi cantl y different from

WKY

Table 2. Characteristics of femoral bone

WKY SHR

Numb er of rats 7 7

Leng th( mm ) 38.1 _+ 0.2 37.0 +_ 0.2 ~

Vo lume (cm 3) 0.67 0.01 0.69 _+ 0.01

Cort ica lth ickn ess ( ) 34.0 0.7 25.6 0.4 b

Dr y weight (rag) 656 10 659 -+ 13

Total as h (mg) 433 -+ 7 418 -+ 12

Ash weight/Volume

(mg/cm3) 650 6 604 _+ 12a

Ash/Dry weight

(Whole bone ) 65.9 0.2 63.3 0.6 a

Ash/Dry weight

(Cor tic al bone) ( ) 67.7 1.2 70.1 _+ 1.0

Values are mean s _+ SE. WKY, normo tens ive Wista r-Kyot o rat;

SHR, spontaneously hypertensive rat

a p < 0.01, bp < 0.00 l--sign ifican tly different from WKY

bone. To test this possibility, we examined the fe-

murs and tibias from SHR and WKY rats chemi-

cally and histomorphologically.

Throughout the period of observation, growth, as

assessed by body weight gain, was similar between

SHR and WKY rats. At 26 weeks of age, mean

systolic blood pressure (SBP) was significantly

greater in SHR than in WKY (Table 1). Serum Ca

levels were similar in SHR and WKY, but serum Pi

levels were significantly decrea sed in SHR. Urinary

excretio ns of Ca were significantly less in SHR than

in WKY, and this may have reflected the lowered

Ca intake due to the decreased food consumption

or the depres sed Ca absorpt ion from the gut in SHR

strain compared with WKY strain [6]. Pi excretions

in the urine were virtually identical for the two

strains.

The morphological measurements and the chem-

Y. Izawa et al.: Bone Disorders in Spontaneously Hypertensive Rat

Table 3. Morphological measurem ents of tibial bone

WKY SHR

Numbe r of rats 5 5

Total bone area (mm2) 7.70 _+ 0.11 8.71 _+: 0.16 a

Cortical area (mm2) 5.09 0.14 5.02 0.24

Medullary area (mm2) 2.61 _+ 0.10 3.69 _: 0.07 a

Cort ica l ar ea ( ) 66.2 _+ 0.9 57.9 0.6 a

Medull ary ar ea ( ) 33.9 -+ 0.9 42.4 -+: 0.4 a

Values are means SE. WKY, normotensive Wistar-Kyoto rat;

SHR, spontaneously hypertensive rat. Percent cortical area and

medullary area were obtained by dividing cortical area or med-

ullary area by total bone area, respectively

a Significantly different from WKY (P < 0.001)

ical compositions of the femurs are given in Table

2; morpholog ical measur ement s of the tibias are

given in Table 3. The femurs of SHR were signifi-

cantly shorter than those of WKY rats, but there

was no difference in the bone volume. The mean

mid-shaft cortical thickness of SHR was reduced

remarkably as compared with WKY. The mean ash

weight per unit bone volume and the ash as per-

centage of dry weight were significantly lower in

SHR than in WKY, although no significant differ-

ence was observed in the dry weight and total ash

weight of the femoral whole bone among the two

strains. On the other hand, in the femoral cortical

bone, the ash as percentage of dry weight was not

significantly different between SHR and WKY

strains. When SHR were compared with WKY, the

total bone area and the medullary area of SHR were

significantly greater, whereas the cortical area was

very similar; consequent ly, the percent cortical area

was significantly lower. In contrast, the percent

medullary area was significantly greater (Table 3).

Microradiographs o f the tibial cross-sections are

shown in Figure 1. In the SH R s tibia, t he medul-

lary area was remarkably greater and consequently

the cortical area was relatively narrower than those

of WKY rats. In addition, an obvious reduction in

trabecular bone mass was observed in SHR. But

neither an increase of bone resorpti on cavity nor a

reduction of bone density on the cortical bone of

SHR was noted as compared with WKY rats.

These results demonstrated that spontaneously

hypertensive rats exhibited reductions of cortical

thickness, longitudinal bone growth, and percent

cortical area compared with normotensive rats. In

addition, femoral analysis showed that no differ-

ence was observed in the ash as percentage of dry

weight of femoral cortical bone between the two

strains, in spite of the reductions of ash as per-

centage of dry weight and ash weight per unit bone

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