Calcif Tissue Int (1985) 37:605-607

Calcified Tissue
International
9 1985 by Springer-Verlag

Bone Disorders in Spontaneously Hypertensive Rat

Yoshihiro Izawa, Kiyoshi Sagara, Takashi Kadota, and Tokutaro Makita
Teijin Institute for Bio-medical Research, 4-3-2 Asahigaoka, Hino-city, Tokyo 191, Japan

Summary. The bones of adult (26 weeks old) spontaneously hypertensive rats (SHR) were examined
chemically and histologically by comparing them
with those of the corresponding normotensive
Wistar-Kyoto (WKY) rats. The mean cortical thickness, the mean ash weight per unit bone volume,
and the ash as percentage of dry weight of femur
were significantly lower in SHR than in WKY. Besides, the percent cortical area measured on tibial
cross-section was also reduced in SHR compared
with WKY. These findings strongly suggest that the
development of osteoporotic bone disorders exists
in adult spontaneously hypertensive rats.
Key words: SHR - - WKY - - Osteoporosis - - Bone.

Introduction
Recent studies have demonstrated that the metabolism of calcium is abnormal in hypertension. However, little attention has been focused on association
between hypertension and bone disorders. In the
present study, therefore, we examined bone tissues
biochemically and histologically in normotensive
and spontaneously hypertensive rats at an age after
the development of hypertensive blood pressure.
The present observations provide evidence that osteoporosis is developed in spontaneously hypertensive rats.
Materials and Methods
Ten male spontaneously hypertensive (SHR) and normotensive
Wistar-Kyoto (WKY) rats were obtained from Nippon CharlesRiver 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% calcium and l% phosphorus with 2,000 units of vitamin D per kg
Send 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 with Rat Tail Manometer-Tachometer System (KN-210, Natsume, 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 anesthesia. Serum Ca and Pi concentrations were determined. Ca
and Pi were 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 plethysmometer (KN-357, Natsume). Roentgenograms were taken with the
use of a Softex (CSM), and from these, cortical thickness of the
femur was measured at the middle of the whole bone and expressed as % of the total width of the bone [1]. They were subsequently dried in an oven at 110~ for 5 days for the measurements of dry weight. Dried bones were ashed in porcelain crucibles 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 content according to the same method described above.
The right tibias were fixed in 70% ethanol and embedded
without decalcification in polyester resin (Ligorac | Nissin EM
Inc.), and three serial cross-sections (50-60 ~xm) per one specimen were cut at the point of one-third from the proximal end
of the bone. Then the sections were examined histomorphologically with contact microradiography (CMR). The tibia was
morphologically measured with an image analyzer [2].
Student's t-test was used to evaluate the significances between
SHR and WKY groups.

Results and Discussion

Recent studies have demonstrated that serum ionized Ca concentrations were reduced [3], serum immunoreactive parathyroid hormone (PTH) levels increased [4], and intestinal Ca absorption reduced in
the spontaneously hypertensive rats (SHR) as compared with those in the normotensive Wistar-Kyoto
rats (WKY) [51. Schedl et al. [5] have also recently
reported that the possibility of abnormal metabolism of vitamin D might exist in SHR. These findings strongly suggest possible abnormalities of

606

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

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

concentration and urinary excretion of calcium and inorganic
phosphorus
WKY
N u m b e r of rats
Body weight (g)
SBP (mmHg)
Serum Ca (mg/100ml)
Serum Pi (mg/100ml)
Urinary Ca (rag/day)
Urinary Pi (rag/day)

SHR

l0
408
160
9.44
5.26
0.78
19.4

14
5
0.05
~ 0.11
0.04
0.9

10
386
236
9.23
4.22
0.54
21.1

+_

24 ~
10c
0.09
0.30 b
0.03 ~
1.1

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

SHR, spontaneously hypertensive rat
a p < 0.05, bp < 0.01, cp < 0.001--significantly different from
WKY

Table 2. Characteristics of femoral bone

WKY
N u m b e r of rats
Length(mm)
Volume (cm 3)
Corticalthickness (%)
Dry weight (rag)
Total ash (mg)
Ash weight/Volume
(mg/cm 3)
Ash/Dry weight
(Whole bone)
Ash/Dry weight
(Cortical bone) (%)

SHR

7
38.1
0.67
34.0
656
433

_+

-+

650

65.9

0.2

63.3

67.7

1.2

70.1

_+ 1.0

0.2
0.01
0.7
10
7

7
37.0
0.69
25.6
659
418

+_
_+

-+
-+

604

_+ 12a

0.2 ~
0.01
0.4 b
13
12

0.6 a

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

SHR, spontaneously hypertensive rat
a p < 0.01, bp < 0.00l--significantly different from WKY

bone. To test this possibility, we examined the femurs and tibias from SHR and WKY rats chemically 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 decreased in SHR. Urinary
excretions 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 depressed Ca absorption 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-

Table 3. Morphological measurements of tibial bone

WKY
N u m b e r of rats
Total bone area (mm 2)
Cortical area (mm 2)
Medullary area (mm 2)
Cortical area (%)
Medullary area (%)

5
7.70
5.09
2.61
66.2
33.9

SHR

_+ 0.11
0.14
_+ 0.10
_+ 0.9
-+ 0.9

5
8.71
5.02
3.69
57.9
42.4

_+: 0.16 a
0.24
_: 0.07 a
0.6 a
-+: 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 medullary area by total bone area, respectively
a Significantly different from W K Y (P < 0.001)

ical compositions of the femurs are given in Table

2; morphological measurements of the tibias are
given in Table 3. The femurs of SHR were significantly 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 percentage of dry weight were significantly lower in
SHR than in WKY, although no significant difference 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 b e t w e e n 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; consequently, the percent cortical area
was significantly lower. In contrast, the percent
medullary area was significantly greater (Table 3).
Microradiographs of the tibial cross-sections are
shown in Figure 1. In the SHR's tibia, the medullary 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 resorption 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 difference 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 percentage of dry weight and ash weight per unit bone

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

607

Fig. 1. Microradiographs of tibial cross-sections from the normotensive Wistar-Kyoto rat (WKY) left, and spontaneously hypertensive
rat (SHR) right.

volume of femoral whole bone. These data strongly

suggest that the development of osteoporotic bone
disorders exists in adult spontaneously hypertensive rats.
These findings may suggest some relationship between alterations of calcuim regulating factors such
as parathyroid hormone and vitamin D 3 metabolites
and development of osteoporosis in spontaneously
hypertensive rats. On the other hand, it seems to
be very important to consider the possibility that
the differences between the two strains of rats represent a genetically derived difference in their responsiveness to the endocrine factors that control
growth generally and the biochemical ones that relate bone mass to mechanical usage. However, the
precise mechanism that causes this bone disorder
in SHR remains obscure, and further studies are
required to clarify the nature and pathogenesis of
the bone disorder in spontaneously hypertensive
rats.

References
1. Orimo H, Fujita T, Yoshikawa M (1971) Effect of calcitonin
on the development of immobilization osteoporosis in rats.
Endocrinol Jpn 18(1): 117-121
2. K o n n o T, Takahashi H (1983) Bone histomorphometry:
image analysis. In: Takahashi H (ed) Handbook of bone morphometry. Nishimura Co., Ltd., Niigata pp 87-89 (Japanese)
3. Wright GL, Rankin GD (1982) Concentrations of ionic and
total calcium in plasma of four models of hypertension. Am
J Physiol 243:H365-H370
4. McCarron DA, Yung NN, Ugorets BA, Krutzik S (1981)
Disturbances of calcium metabolism in the spontaneously
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(1984) Calcium and sodium transport and vitamin D metabolism in the spontaneously hypertensive rat. J Clin Invest
73:980-986
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