Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 305, 123-126 (1978)
Archivesof
Pharmacology 9 by Springer-Verlag 1978.
Chronic Hypertension Induced by Streptozotocin in Rats H i d e t o s h i K a w a s h i m a , Toshiji Igarashi, Y o s h i k a g e N a k a j i m a , Y a s u h i r o A k i y a m a , K a z u y a s u U s u k i , and Shinzaburo Ohtake Department of Pharmacology, Section of Experimental Therapeutics Research, Eisai Co., Ltd., Tokyo 112, Japan
Summary. A n i n t r a v e n o u s injection o f 40 or 65 m g / k g s t r e p t o z o t o c i n i n d u c e d n o t o n l y d i a b e t e s b u t also severe h y p e r t e n s i o n i n rats. W h e r e a s the h y p e r g l y c e m i a d e v e l o p e d fully within a few d a y s after the injection o f s t r e p t o z o t o c i n , the h y p e r t e n s i o n progressively adv a n c e d a n d r e a c h e d m a x i m u m level several weeks after the t r e a t m e n t a n d lasted m o r e t h a n 20 weeks. T w e n t y m g / k g s t r e p t o z o t o c i n d i d n o t induce h y p e r g l y c e m i a b u t significantly increased b l o o d pressure several weeks after the t r e a t m e n t . A r r e s t o f growth, p o l y u r i a , glycosuria, h y p e r l i p e m i a a n d lenticular c a t a r a c t s d e v e l o p e d in the a n i m a l s t r e a t e d with 40 o r 65 m g / k g s t r e p t o z o tocin, b u t in n o n e o f the a n i m a l s t r e a t e d with 20 mg/kg. In histological e x a m i n a t i o n s in the 24th week after the t r e a t m e n t , d e g r a n u l a t i o n a n d necrosis in the p a n c r e a t i c fl-cells, a n d v a c u o l i z a t i o n a n d d e p o s i t i o n o f P A S positive m a t e r i a l s in the renal p r o x i m a l tubules were f o u n d in the a n i m a l s t r e a t e d with 40 or 6 5 m g / k g streptozotocin. Key words: B l o o d p r e s s u r e Cataract -
B l o o d glucose
-
Glycosuria.
Introduction It is generally k n o w n t h a t m a n y d i a b e t i c p a t i e n t s suffer f r o m m i l d o r severe h y p e r t e n s i o n , a l t h o u g h the rel a t i o n s h i p between d i a b e t e s mellitus a n d h y p e r t e n s i o n is n o t well defined ( G o l d e n b e r g et al., 1958; Pell a n d D ' A l o n z o , 1967). S t r e p t o z o t o c i n is a n a n t i b i o t i c obt a i n e d f r o m Streptomyces achromogenes ( H e r r et al., 1967). It is well e s t a b l i s h e d t h a t this agent induces h y p e r g l y c e m i a in rats ( R a k i e t e n et al., 1963; Schein et al., 1967) a n d in o t h e r a n i m a l species. T h e d i a b e t o g e n i c effect o f s t r e p t o z o t o c i n is caused b y irreversible d a m a g e
Send offprint requests to H. Kawashima at the above address
to p a n c r e a t i c fl-cells ( A r i s o n et al., 1967; J u n o d et al., 1967). T h e p r e s e n t p a p e r r e p o r t s t h a t a single injection o f s t r e p t o z o t o c i n induces n o t o n l y d i a b e t e s b u t also l o n g - l a s t i n g h y p e r t e n s i o n in rats.
Materials and Methods Experiments were carried out with male Sprague-Dawley rats weighing 210-250 g. The animals were fed a commercially prepared pellet diet (CE-2, Japan CLEA Co., Tokyo) and tap water ad libitum. Sixty animals were randomly divided into 3 groups of 20 animals, which were treated with an intravenous injection of 20, 40 or 65mg/kg streptozotocin. An additional group of 7non-treated animals served as the normal controls. Streptozotocin (Sigma) was dissolved in 0.9 % saline containing I mM citrate buffer (pH 4.5) immediately before use. The prepared solution was injected into the superficial cutaneous vein of the foot at a volume of 0.1 ml per 100g body weight. Blood (0.03 ml) was taken from the tail vein and blood glucose was measured according to the anthrone method (Ui, 1965). Urine was collected for 4 h using a metabolic cage and urinary glucose was determined by the enzyme method using a glucose analyzer (Morrison, 1972). For the determination of blood pressure, rats were restrained in plastic holders (KN-325 (B), Natsume Seisaku-sho Co., Tokyo). Blood pressure and heart rate were determined by a tail plethysmography cuff method using the continuous systolic monitor (SCS-301, Shimadsu Seisaku-sho Co., Tokyo) in a warmed box (30~C). The apparatus contains an inflatable occluding cuff and two photoelectric sphygmographs. Two pulse detectors of the sphygmographies are set on the tail proximal and distal to the occluding cuff. Air pressure in the annular cuff is automatically adjusted at the critical level where sphygmograms led from both detectors are able to synchronize. Heart rate was measured with a tachometer which was triggered by the sphygmogram from the proximal detector (Igarashi et al., 1974, 1977). In the 24th week after the injection of streptozotocin, 7 animals randomly selected from each group were killed by decapitation and blood samples collected. After centrifugation at 1055 x g for 10min, a serum sample was taken and stored at -20~ until assayed. The remaining rats were kept for further observation. Serum triglyceride was determined by the acetylacetone method (Fletcher, 1968), NEFA by the method of Laurell and Tibbling (1967), fl-lipoprotein by the method of Fried and Hoeflmayr (1963), total cholesterol by the method of Altain et al. (1974), BUN by the diacetylmonoxime method (Ormsby, 1942), lactate by the method of Marbach and Weil
0028-1298/78/0305/0123/801.00
124
Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) 10(3 INCIDENCE OF CATARACTS(%)
BLOOD GLUCOSE (mgldl)
65 mg/kg
700
} (13./17)
600 513
500
/
400
~
--
__l
40 mg/kg
"r
I
1(4/20)
300
C~
200 ............ ,
b
-
() -
,4
12
0
4
8
12
16
20
6mg,kg
.
BLOOD PRESSURE (rnmHg)
220 200
2~0
~
100
0
40 mg/kg
1006
65 mg/kg /.~....~"------------~
URINARY GLUCOSE
100
180 160 140
.........
o ...............
.......
20 mg/kg
~
o
120
4
~
12
1~
2'o -
CONTROL
100. 0
4
9 12 16 20 WEEKS AFTER STREPTOZOTOCIN
Fig. 1. Changes in blood glucose (upper graph) and blood pressure (lower graph) during 20 weeks after an intravenous injection of 20, 40 or 65 mg/kg streptozotocin. Each point represents the mean of values obtained from 7 to 20 animals, and the vertical bars indicate S.E.M.
500
BODY
~
"
-
~
"
Results Figure i shows changes in blood glucose and blood pressure during 20 weeks after an intravenous injection of streptozotocin. On the 3rd day, a marked hyper 7 glycemia was found in all animals treated with 40 or 65 mg/kg streptozotocin, whereas blood glucose level in the animals treated with 20 mg/kg .. was in the normal range. On the 4th day, blood pressure was determined in 7 animals randomly selected from each group. Mean values of blood pressure in the groups of 20, 40 and 65 mg/kg were 127 + 4, 144 + 5 and 159 + 6 mm Hg, respectively. The mean value of blood pressure in the controls was 124 _+ 3 mm Hg. Blood pressure in all the animals treated with streptozotocin progressively increased thereafter and reached maximum level 4--12 weeks after the treatment: in the 8th week after the
....
I
20 mg/kg
300~ ~ ~ 6~5mg/kg
200
(1967), sodium and potassium by a flame photometer, chloride by Evans chloride meter, calcium by the o-cresolphthalein complexon method (Morin, 1974) and magnesium by the method of Rice and Lapara (1964). On autopsy, organs (pancreas, kidneys, adrenals, liver and- brain) were fixed in 10 % neutral formalin solution and embedded in paraffin for light microscopy. Pancreatic sections were stained with Victoriablue for the identification of the islet B-cells, and the others were stained with hematoxylin and eosin (H&E), and periodic acid-Schiff reagents (PAS). All results in Figures were expressed as mean _+ standard error of the mean (S.E.M.). Data were analyzed by the method described by Taguchi (1972), and a pvalue of less than 0.05 was considered significant.
~
~ - ~ - - ~
WEIGHT /g)
~
400
~
t
,~.
8
12
16
20
WEEKS AFTER STREPTOZOTOCIN Fig. 2. Body weight, urinary glucose excretion and incidence o f cataracts after an intravenous injection of 20, 40 or 65mg/,kg streptozotocin, l~ach point represents the mean of values obtained from 7 to 20 animals with S.E.M.
treatment, moderate hypertension above 160mm Hg was induced in 17 of 20 animals treated with 20 mg/kg, severe hypertension exceeding 200 mm Hg developed in 8 of 20 animals treated with 40mg/kg and in 14 of 18 animals treated with 65 mg/kg. Blood pressure in the normal controls fluctuated in a range of 1 2 0 140mmHg during the period of the experiment. Heart rate was in a range of 320-360 beats/rain at the beginning of the experiment and tended to decrease to 300-330 beats/rain in several weeks. No significant differences in the mean values of heart rate were found among the groups. Figure 2 shows the amount of glucose excreted into the urine, body weight and development of lenticular cataracts. The amount of glucose excreted into urine was determined 0, 1, 3, 6, 12 and 18weeks after treatment with streptozotocin. Glycosuria was not found in any animals in the group treated with 20mg/kg, whereas a various amount of urinary glucose was detected in all animals at the dose of 40 or 65 mg/kg. In contrast to the hyperglycemia which reached maximum level within a few days after treatment with streptozo-
H. Kawashima et al. : Streptozotocin-Induced Hypertension
tocin, the urinary glucose progressively increased until 12 weeks after the treatment. Arrest of growth was one of prominent toxic signs due to streptozotocin, although the animals in the group of 20 mg/kg had a normal weight gain during the 20 weeks of the experiment. Cataracts developed in the animals treated with 40 or 65 mg/kg (Fig.2) : in the 16th week after the treatment lenticular opacities were grossly found in 4 of 19 animals treated with 40 mg/kg, and in 15 of 17 animals treated with 65 mg/kg, but in none of the 20 rats treated with 20 mg/kg. Besides a marked hyperglycemia induced by streptozotocin, definite increases in serum cholesterol, triglyceride and BUN, and decreases in serum sodium and chloride were found in the animals treated with 40 or 65mg/kg at the 24th week after the injection of streptozotocin. The levels of all serum metabolites examined in the animals at the dose level of 20 mg/kg were in the range of controls. Other remarkable toxic signs in animals treated with 40 or 65mg/kg streptozotocin were polyuria, polydipsia and polyphagia. One of 20 animals treated with 40mg/kg and 4 of 20animals treated with 65mg/kg, but none of the 20animals treated with 20 mg/kg died during the 20 weeks following treatment with streptozotocin. Histologic examination in the 24th week after the treatment revealed that fi-cell necrosis and degranulation of surviving/%cells in the islet were seen in all animals treated with 40 or 65 mg/kg, but in none of the animals treated with 20mg/kg. In the renal tissue, vacuolization and deposition of PAS-positive materials in the proximal tubular epithelial cells were found in all animals treated with 65 mg/kg, and 4 of 7 animals treated with 40 mg/kg but in none of the animals treated with 20 mg/kg. No remarkable changes in the renal glomerular apparatus, adrenals, liver, heart and brain were found in any groups.
125
response several weeks after the administration of streptozotocin. The lowest dose level, 20 mg/kg did not affect the blood glucose level but significantly increased the blood pressure. These facts indicate that hyperglycemia is not a prerequisite for induction of hypertention by streptozotocin. The data, however, would not exclude the possibility that the hypertensive action of streptozotocin is secondary to the suppression of insulin secretion. The degree of glycosuria markedly increased in progress of time after the treatment with diabetogenic doses, and the amount of urinary glucose at the 1st week after the treatment increased up to about 100 times at the 12th week. On the histological examinations on the kidney at the 24th week, vacuolization and accumulation of PAS-positive materials in the proximal tubular epithelial cells were found in the rats treated with 40 or 65 mg/kg. Although the progression of glycosuria, increased serum BUN and such pathological changes found in the renal tubular cells are regarded as signs of renal dysfunction, they appear to be associated with the long-term diabetic state rather than any direct effects of this agent on the renal tissue. According to the light and electron microscopical studies by Arison et al. (1967), notable changes in the kidney of rats treated with a diabetogenic dose of streptozotocin were heavy deposition of glycogen in the proximal tubules and slight thickening of the glomerular basement membrane. They concluded that the direct and immediate action of streptozotocin is specifically on the pancreatic /3-cells. The results of the present study demonstrated that an intravenous injection of streptozotocin induced not only diabetes but also chronic hypertension in rats. However, the mechanism of hypertension induced by streptozotocin remains to be solved.
References Discussion
Streptozotocin has received much attention since Rakieten et al., reported its diabetogenic effects in 1963. However, as far as we know, there has been no evidence which suggests the hypertensive action itself or any pharmacological features relating the mechanism of hypertensive action of this agent. The mechanism of diabetogenic action of streptozotocin is generally believed to be the consequence of degranulation of the fl-cells, resulting in loss of insulin secretion (Junod et al., 1967; Stauffacher et al., 1970). In the present study, blood glucose increased to the maximum level within a few days, but blood pressure progressively increased and reached to the maximum
Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W., Fu, P. C. : Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470-475 (1974) Arison, R. N., Ciaccio, E. I., Glitzer, M. S., Cassaro, J. A., Pruss, M. P. : Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin. Diabetes 16, 5 1 - 56 (1967) Fletcher, M. J. : A colorimetric method for estimation of sermn triglycerides. Ctin. Chim. Acta 22, 393-~ 397 (1968) Fried, R., Hoeflmayr, J. : Eine einfache indirekte Bestimmungsmethode der/~-Lipoproteide. Klin. Wochenschr. 41, 246 (1963) Goldenberg, S., Alex, M., Blumenthal, H. T.: Sequelae of arteriosclerosis of the aorta and coronary arteries. Diabetes 7, 98-108
0958) Herr, R. R., Jahnku, H. K., Argondelis, A. D.: The structure of streptozotocin. J. Am. Chem. Soc. 89, 4808-4809 (1967) Igarashi, T., Nakajima, Y., Tanaka, M , Ohtake, S.: Effect of Coenzyme Q,0 on experimental hypertension in rats and dogs. J. Pharmacol. Exp. Ther. 189, 149-156 (1974)
126 Igarashi, T., Nakajima, Y., Ohtake, S. : Antihypertensive effect of combined treatment with ct- and fl-adrenergic blockers in the Spontaneously Hypertensive Rat. Jpn. Circ. J. 41, 903-911 (1977) Junod, A., Lambert, A. E., Orci, L., Pictet, R., Gonet, A. E., Renold, A. E. : Studies of the diabetogenic action of streptozotocin. Proc. Soc. Exp. Biol. Med. 126, 201-205 (1967) Laurell, S., Tibbling, G. : Colorimetric microdetermination of free fatty acids in plasma. Clin. Chim. Acta 16, 5 7 - 6 2 (1967) Marbach, E. P., Weil, M. H.: Rapid enzymatic measurement of blood lactate and pyruvate. Clin. Chem. 13, 314-325 (1967) Morin, L. G. : Direct colorimetric determination of serum calcium with o-eresophthale~ complexon. Am. J. Clin. Path. 61, 114117 (1974) Morrison, B. : Use of the Beckman Glucose Analyzer for low and high glucose values. Clin. Chim. Acta 42, 192 (1972) Ormsby, A. A. : A direct colorimetric method for the determination of urea in blood and urine. J. Biol. Chem. 146, 595-604 (1942) Pell, S., D'Alonzo, C. A. : Some aspects of hypertension in diabetes mellitus. J. Am. Med. Assoc. 202, 10-16 (1967)
Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) Rakieten, N., Rakieten, M. L., Nadkarni, M. V. : The diabetogenic action of streptozotocin. Cancer Chemother. Rep. 29, 91-98 (1963) Rice, E. W., Lapara, C. Z.: Rapid ultramicrospectrophotometric determination of magnesium. Clin. Chim. Acta I0, 360-364 (1964) Schein, P. S., Cooney, D. A., Vernon, M. L. : The use of nicotinamide to modify the toxicity of streptozotocin diabetes without loss of antitumor activity. Cancer Res. 27, 2324-2332 (1967) Stauffacher, W., Burr, I., Gutzeit, A. Beaven, D., Veleminsky, J , Renold, A. E. : Streptozotocin diabetes: Time course of irreversible B-~II damage; Further observation on prevention by nicotinamide. Proc. Soc. Exp. Biol. Med. 133, 194-200 (1970) Taguchi, G.: Statistical analysis method, pp. 21-54. Tokyo: Maruzen 1972 Ui, M.: Blockage of epinephrine-induced hyperglycemia during exposure to simulated altitudes. Am. J. Physiol. 209, 353--358 (1965)
Received June 6/Accepted August 28, 1978
Archivesof
Pharmacology 9 by Springer-Verlag 1978.
Chronic Hypertension Induced by Streptozotocin in Rats H i d e t o s h i K a w a s h i m a , Toshiji Igarashi, Y o s h i k a g e N a k a j i m a , Y a s u h i r o A k i y a m a , K a z u y a s u U s u k i , and Shinzaburo Ohtake Department of Pharmacology, Section of Experimental Therapeutics Research, Eisai Co., Ltd., Tokyo 112, Japan
Summary. A n i n t r a v e n o u s injection o f 40 or 65 m g / k g s t r e p t o z o t o c i n i n d u c e d n o t o n l y d i a b e t e s b u t also severe h y p e r t e n s i o n i n rats. W h e r e a s the h y p e r g l y c e m i a d e v e l o p e d fully within a few d a y s after the injection o f s t r e p t o z o t o c i n , the h y p e r t e n s i o n progressively adv a n c e d a n d r e a c h e d m a x i m u m level several weeks after the t r e a t m e n t a n d lasted m o r e t h a n 20 weeks. T w e n t y m g / k g s t r e p t o z o t o c i n d i d n o t induce h y p e r g l y c e m i a b u t significantly increased b l o o d pressure several weeks after the t r e a t m e n t . A r r e s t o f growth, p o l y u r i a , glycosuria, h y p e r l i p e m i a a n d lenticular c a t a r a c t s d e v e l o p e d in the a n i m a l s t r e a t e d with 40 o r 65 m g / k g s t r e p t o z o tocin, b u t in n o n e o f the a n i m a l s t r e a t e d with 20 mg/kg. In histological e x a m i n a t i o n s in the 24th week after the t r e a t m e n t , d e g r a n u l a t i o n a n d necrosis in the p a n c r e a t i c fl-cells, a n d v a c u o l i z a t i o n a n d d e p o s i t i o n o f P A S positive m a t e r i a l s in the renal p r o x i m a l tubules were f o u n d in the a n i m a l s t r e a t e d with 40 or 6 5 m g / k g streptozotocin. Key words: B l o o d p r e s s u r e Cataract -
B l o o d glucose
-
Glycosuria.
Introduction It is generally k n o w n t h a t m a n y d i a b e t i c p a t i e n t s suffer f r o m m i l d o r severe h y p e r t e n s i o n , a l t h o u g h the rel a t i o n s h i p between d i a b e t e s mellitus a n d h y p e r t e n s i o n is n o t well defined ( G o l d e n b e r g et al., 1958; Pell a n d D ' A l o n z o , 1967). S t r e p t o z o t o c i n is a n a n t i b i o t i c obt a i n e d f r o m Streptomyces achromogenes ( H e r r et al., 1967). It is well e s t a b l i s h e d t h a t this agent induces h y p e r g l y c e m i a in rats ( R a k i e t e n et al., 1963; Schein et al., 1967) a n d in o t h e r a n i m a l species. T h e d i a b e t o g e n i c effect o f s t r e p t o z o t o c i n is caused b y irreversible d a m a g e
Send offprint requests to H. Kawashima at the above address
to p a n c r e a t i c fl-cells ( A r i s o n et al., 1967; J u n o d et al., 1967). T h e p r e s e n t p a p e r r e p o r t s t h a t a single injection o f s t r e p t o z o t o c i n induces n o t o n l y d i a b e t e s b u t also l o n g - l a s t i n g h y p e r t e n s i o n in rats.
Materials and Methods Experiments were carried out with male Sprague-Dawley rats weighing 210-250 g. The animals were fed a commercially prepared pellet diet (CE-2, Japan CLEA Co., Tokyo) and tap water ad libitum. Sixty animals were randomly divided into 3 groups of 20 animals, which were treated with an intravenous injection of 20, 40 or 65mg/kg streptozotocin. An additional group of 7non-treated animals served as the normal controls. Streptozotocin (Sigma) was dissolved in 0.9 % saline containing I mM citrate buffer (pH 4.5) immediately before use. The prepared solution was injected into the superficial cutaneous vein of the foot at a volume of 0.1 ml per 100g body weight. Blood (0.03 ml) was taken from the tail vein and blood glucose was measured according to the anthrone method (Ui, 1965). Urine was collected for 4 h using a metabolic cage and urinary glucose was determined by the enzyme method using a glucose analyzer (Morrison, 1972). For the determination of blood pressure, rats were restrained in plastic holders (KN-325 (B), Natsume Seisaku-sho Co., Tokyo). Blood pressure and heart rate were determined by a tail plethysmography cuff method using the continuous systolic monitor (SCS-301, Shimadsu Seisaku-sho Co., Tokyo) in a warmed box (30~C). The apparatus contains an inflatable occluding cuff and two photoelectric sphygmographs. Two pulse detectors of the sphygmographies are set on the tail proximal and distal to the occluding cuff. Air pressure in the annular cuff is automatically adjusted at the critical level where sphygmograms led from both detectors are able to synchronize. Heart rate was measured with a tachometer which was triggered by the sphygmogram from the proximal detector (Igarashi et al., 1974, 1977). In the 24th week after the injection of streptozotocin, 7 animals randomly selected from each group were killed by decapitation and blood samples collected. After centrifugation at 1055 x g for 10min, a serum sample was taken and stored at -20~ until assayed. The remaining rats were kept for further observation. Serum triglyceride was determined by the acetylacetone method (Fletcher, 1968), NEFA by the method of Laurell and Tibbling (1967), fl-lipoprotein by the method of Fried and Hoeflmayr (1963), total cholesterol by the method of Altain et al. (1974), BUN by the diacetylmonoxime method (Ormsby, 1942), lactate by the method of Marbach and Weil
0028-1298/78/0305/0123/801.00
124
Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) 10(3 INCIDENCE OF CATARACTS(%)
BLOOD GLUCOSE (mgldl)
65 mg/kg
700
} (13./17)
600 513
500
/
400
~
--
__l
40 mg/kg
"r
I
1(4/20)
300
C~
200 ............ ,
b
-
() -
,4
12
0
4
8
12
16
20
6mg,kg
.
BLOOD PRESSURE (rnmHg)
220 200
2~0
~
100
0
40 mg/kg
1006
65 mg/kg /.~....~"------------~
URINARY GLUCOSE
100
180 160 140
.........
o ...............
.......
20 mg/kg
~
o
120
4
~
12
1~
2'o -
CONTROL
100. 0
4
9 12 16 20 WEEKS AFTER STREPTOZOTOCIN
Fig. 1. Changes in blood glucose (upper graph) and blood pressure (lower graph) during 20 weeks after an intravenous injection of 20, 40 or 65 mg/kg streptozotocin. Each point represents the mean of values obtained from 7 to 20 animals, and the vertical bars indicate S.E.M.
500
BODY
~
"
-
~
"
Results Figure i shows changes in blood glucose and blood pressure during 20 weeks after an intravenous injection of streptozotocin. On the 3rd day, a marked hyper 7 glycemia was found in all animals treated with 40 or 65 mg/kg streptozotocin, whereas blood glucose level in the animals treated with 20 mg/kg .. was in the normal range. On the 4th day, blood pressure was determined in 7 animals randomly selected from each group. Mean values of blood pressure in the groups of 20, 40 and 65 mg/kg were 127 + 4, 144 + 5 and 159 + 6 mm Hg, respectively. The mean value of blood pressure in the controls was 124 _+ 3 mm Hg. Blood pressure in all the animals treated with streptozotocin progressively increased thereafter and reached maximum level 4--12 weeks after the treatment: in the 8th week after the
....
I
20 mg/kg
300~ ~ ~ 6~5mg/kg
200
(1967), sodium and potassium by a flame photometer, chloride by Evans chloride meter, calcium by the o-cresolphthalein complexon method (Morin, 1974) and magnesium by the method of Rice and Lapara (1964). On autopsy, organs (pancreas, kidneys, adrenals, liver and- brain) were fixed in 10 % neutral formalin solution and embedded in paraffin for light microscopy. Pancreatic sections were stained with Victoriablue for the identification of the islet B-cells, and the others were stained with hematoxylin and eosin (H&E), and periodic acid-Schiff reagents (PAS). All results in Figures were expressed as mean _+ standard error of the mean (S.E.M.). Data were analyzed by the method described by Taguchi (1972), and a pvalue of less than 0.05 was considered significant.
~
~ - ~ - - ~
WEIGHT /g)
~
400
~
t
,~.
8
12
16
20
WEEKS AFTER STREPTOZOTOCIN Fig. 2. Body weight, urinary glucose excretion and incidence o f cataracts after an intravenous injection of 20, 40 or 65mg/,kg streptozotocin, l~ach point represents the mean of values obtained from 7 to 20 animals with S.E.M.
treatment, moderate hypertension above 160mm Hg was induced in 17 of 20 animals treated with 20 mg/kg, severe hypertension exceeding 200 mm Hg developed in 8 of 20 animals treated with 40mg/kg and in 14 of 18 animals treated with 65 mg/kg. Blood pressure in the normal controls fluctuated in a range of 1 2 0 140mmHg during the period of the experiment. Heart rate was in a range of 320-360 beats/rain at the beginning of the experiment and tended to decrease to 300-330 beats/rain in several weeks. No significant differences in the mean values of heart rate were found among the groups. Figure 2 shows the amount of glucose excreted into the urine, body weight and development of lenticular cataracts. The amount of glucose excreted into urine was determined 0, 1, 3, 6, 12 and 18weeks after treatment with streptozotocin. Glycosuria was not found in any animals in the group treated with 20mg/kg, whereas a various amount of urinary glucose was detected in all animals at the dose of 40 or 65 mg/kg. In contrast to the hyperglycemia which reached maximum level within a few days after treatment with streptozo-
H. Kawashima et al. : Streptozotocin-Induced Hypertension
tocin, the urinary glucose progressively increased until 12 weeks after the treatment. Arrest of growth was one of prominent toxic signs due to streptozotocin, although the animals in the group of 20 mg/kg had a normal weight gain during the 20 weeks of the experiment. Cataracts developed in the animals treated with 40 or 65 mg/kg (Fig.2) : in the 16th week after the treatment lenticular opacities were grossly found in 4 of 19 animals treated with 40 mg/kg, and in 15 of 17 animals treated with 65 mg/kg, but in none of the 20 rats treated with 20 mg/kg. Besides a marked hyperglycemia induced by streptozotocin, definite increases in serum cholesterol, triglyceride and BUN, and decreases in serum sodium and chloride were found in the animals treated with 40 or 65mg/kg at the 24th week after the injection of streptozotocin. The levels of all serum metabolites examined in the animals at the dose level of 20 mg/kg were in the range of controls. Other remarkable toxic signs in animals treated with 40 or 65mg/kg streptozotocin were polyuria, polydipsia and polyphagia. One of 20 animals treated with 40mg/kg and 4 of 20animals treated with 65mg/kg, but none of the 20animals treated with 20 mg/kg died during the 20 weeks following treatment with streptozotocin. Histologic examination in the 24th week after the treatment revealed that fi-cell necrosis and degranulation of surviving/%cells in the islet were seen in all animals treated with 40 or 65 mg/kg, but in none of the animals treated with 20mg/kg. In the renal tissue, vacuolization and deposition of PAS-positive materials in the proximal tubular epithelial cells were found in all animals treated with 65 mg/kg, and 4 of 7 animals treated with 40 mg/kg but in none of the animals treated with 20 mg/kg. No remarkable changes in the renal glomerular apparatus, adrenals, liver, heart and brain were found in any groups.
125
response several weeks after the administration of streptozotocin. The lowest dose level, 20 mg/kg did not affect the blood glucose level but significantly increased the blood pressure. These facts indicate that hyperglycemia is not a prerequisite for induction of hypertention by streptozotocin. The data, however, would not exclude the possibility that the hypertensive action of streptozotocin is secondary to the suppression of insulin secretion. The degree of glycosuria markedly increased in progress of time after the treatment with diabetogenic doses, and the amount of urinary glucose at the 1st week after the treatment increased up to about 100 times at the 12th week. On the histological examinations on the kidney at the 24th week, vacuolization and accumulation of PAS-positive materials in the proximal tubular epithelial cells were found in the rats treated with 40 or 65 mg/kg. Although the progression of glycosuria, increased serum BUN and such pathological changes found in the renal tubular cells are regarded as signs of renal dysfunction, they appear to be associated with the long-term diabetic state rather than any direct effects of this agent on the renal tissue. According to the light and electron microscopical studies by Arison et al. (1967), notable changes in the kidney of rats treated with a diabetogenic dose of streptozotocin were heavy deposition of glycogen in the proximal tubules and slight thickening of the glomerular basement membrane. They concluded that the direct and immediate action of streptozotocin is specifically on the pancreatic /3-cells. The results of the present study demonstrated that an intravenous injection of streptozotocin induced not only diabetes but also chronic hypertension in rats. However, the mechanism of hypertension induced by streptozotocin remains to be solved.
References Discussion
Streptozotocin has received much attention since Rakieten et al., reported its diabetogenic effects in 1963. However, as far as we know, there has been no evidence which suggests the hypertensive action itself or any pharmacological features relating the mechanism of hypertensive action of this agent. The mechanism of diabetogenic action of streptozotocin is generally believed to be the consequence of degranulation of the fl-cells, resulting in loss of insulin secretion (Junod et al., 1967; Stauffacher et al., 1970). In the present study, blood glucose increased to the maximum level within a few days, but blood pressure progressively increased and reached to the maximum
Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W., Fu, P. C. : Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470-475 (1974) Arison, R. N., Ciaccio, E. I., Glitzer, M. S., Cassaro, J. A., Pruss, M. P. : Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin. Diabetes 16, 5 1 - 56 (1967) Fletcher, M. J. : A colorimetric method for estimation of sermn triglycerides. Ctin. Chim. Acta 22, 393-~ 397 (1968) Fried, R., Hoeflmayr, J. : Eine einfache indirekte Bestimmungsmethode der/~-Lipoproteide. Klin. Wochenschr. 41, 246 (1963) Goldenberg, S., Alex, M., Blumenthal, H. T.: Sequelae of arteriosclerosis of the aorta and coronary arteries. Diabetes 7, 98-108
0958) Herr, R. R., Jahnku, H. K., Argondelis, A. D.: The structure of streptozotocin. J. Am. Chem. Soc. 89, 4808-4809 (1967) Igarashi, T., Nakajima, Y., Tanaka, M , Ohtake, S.: Effect of Coenzyme Q,0 on experimental hypertension in rats and dogs. J. Pharmacol. Exp. Ther. 189, 149-156 (1974)
126 Igarashi, T., Nakajima, Y., Ohtake, S. : Antihypertensive effect of combined treatment with ct- and fl-adrenergic blockers in the Spontaneously Hypertensive Rat. Jpn. Circ. J. 41, 903-911 (1977) Junod, A., Lambert, A. E., Orci, L., Pictet, R., Gonet, A. E., Renold, A. E. : Studies of the diabetogenic action of streptozotocin. Proc. Soc. Exp. Biol. Med. 126, 201-205 (1967) Laurell, S., Tibbling, G. : Colorimetric microdetermination of free fatty acids in plasma. Clin. Chim. Acta 16, 5 7 - 6 2 (1967) Marbach, E. P., Weil, M. H.: Rapid enzymatic measurement of blood lactate and pyruvate. Clin. Chem. 13, 314-325 (1967) Morin, L. G. : Direct colorimetric determination of serum calcium with o-eresophthale~ complexon. Am. J. Clin. Path. 61, 114117 (1974) Morrison, B. : Use of the Beckman Glucose Analyzer for low and high glucose values. Clin. Chim. Acta 42, 192 (1972) Ormsby, A. A. : A direct colorimetric method for the determination of urea in blood and urine. J. Biol. Chem. 146, 595-604 (1942) Pell, S., D'Alonzo, C. A. : Some aspects of hypertension in diabetes mellitus. J. Am. Med. Assoc. 202, 10-16 (1967)
Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) Rakieten, N., Rakieten, M. L., Nadkarni, M. V. : The diabetogenic action of streptozotocin. Cancer Chemother. Rep. 29, 91-98 (1963) Rice, E. W., Lapara, C. Z.: Rapid ultramicrospectrophotometric determination of magnesium. Clin. Chim. Acta I0, 360-364 (1964) Schein, P. S., Cooney, D. A., Vernon, M. L. : The use of nicotinamide to modify the toxicity of streptozotocin diabetes without loss of antitumor activity. Cancer Res. 27, 2324-2332 (1967) Stauffacher, W., Burr, I., Gutzeit, A. Beaven, D., Veleminsky, J , Renold, A. E. : Streptozotocin diabetes: Time course of irreversible B-~II damage; Further observation on prevention by nicotinamide. Proc. Soc. Exp. Biol. Med. 133, 194-200 (1970) Taguchi, G.: Statistical analysis method, pp. 21-54. Tokyo: Maruzen 1972 Ui, M.: Blockage of epinephrine-induced hyperglycemia during exposure to simulated altitudes. Am. J. Physiol. 209, 353--358 (1965)
Received June 6/Accepted August 28, 1978
