Renin-angiotensin system in stroke-prone spontaneously hypertensive rats MASAKI SHIBOTA, AKINOBU NAGAOKA, AK10 SHINO, AND TAKESHI FUJITA Biological Research Laboratories, Central Research Division, Taheda Chemical Industries, Osaka 532, Japan
SHIBOTA, MASAKI, AKINOBU NAGAOKA, AKIO SHINO, AND TAKESHI FUJITA. Renin-angiotensin system in stroke-prone spontaneously hypertensive rats. Am. J. Physiol. 236(3): H409H416, 1979 or Am. J. Physiol: Heart Circ. Physiol. 5(3): H409H416, 1979.-The development of malignant hypertension was studied in stroke-prone spontaneously hypertensive rats (SHR) kept on 1% NaCl as drinking water. Along with salt-loading, blood pressure gradually increased and reached a severe hypertensive level (>230 mmHg), which was followed by increases in urinary protein (X00 (mg/250 g body wt)/day) and plasma renin concentration (PRC, from 18.9 t 0.1 to 51.2 & 19.4 (ng/ ml)/h, mean t SD). At this stage, renal small arteries and arterioles showed severe sclerosis and fibrinoid necrosis. Stroke was observed within a week after the onset of these renal abnormalities. The dose of exogenous angiotensin II (AII) producing 30 mmHg rise in blood pressure increased with the elevation of PRC, from 22 t, 12 to 75 t 36 rig/kg, which was comparable to that in rats on water. The fall of blood pressure due to an AI1 inhibitor, [1-sarcosine, &alanine]AII (10 (pg/kg)/ min for 40 min) became more prominent with the increase in PRC in salt-loaded rats, but was not detected in rats on water. These findings suggest that the activation of renin-angiotensin system participates in malignant hypertension of salt-loaded stroke-prone SHR rats that show stroke signs, proteinuria, hyperreninemia, and renovascular changes. salt-loading; urinary lignant hypertension; inhibitor
protein; plasma renin concentration; maresponse to angiotensin II; angiotensin II
SPONTANEOUSLY HYPERTENSIVE RATS (SHR)have been used as a model of human hypertension. Recently, Okamoto et al. (34) established a substrain (stroke-prone SHR rats) from the SHR rats; these rats were more hypertensive and developed cerebrovascular lesions (CVL) with a higher incidence than the original strain. Both the development of hypertension and onset of CVL of stroke-prone SHR rats were accelerated by salt loading (33,34). When the rats were kept on 1% NaCl as drinking water at the age of about 10 wk, most of them showed severe hypertension (>220 mmHg) and developed CVL within 5 wk. These reports suggested that the salt-loaded stroke-prone SHR is an excellent model for studies of malignant hypertension. Proteinuria is usually used as a clinical index in human hypertension (9, 14,25,35, 39). In the malignant phase of hypertension, proteinuria (24, 30) and hyperreninemia 0363-6135/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
(2, 5, 18, 19, 29) are often observed, although some investigators reported some casesof malignant hypertension without hyperreninemia (2, 22, 29). Similarly in animals, the activation of the renin-angiotensin system was reported in the malignant phase of experimental renovascular (3, 26, 31, 32, 36) or spontaneous (28, 43) hypertension. The present study was designed to correlate changes in excretion of urinary protein and plasma renin concentration (PRC) with changes in blood pressure and malignant vascular changes in stroke-prone SHR during prolonged administration of 1% NaCl solution. Roles of the renin-angiotensin system were also examined in the malignant phase hypertension. METHODS
Animals. Experiments were performed on male strokeprone SHR. They were maintained on a laboratory chow, CA-l, containing 10.4 meq of sodium/l00 g (Japan Clea, Tokyo) and water ad libitum. Salt loading was started from about 11 wk of age by giving 1% NaCl as drinking water. Individual metabolic cageswere used for collection of urine. The systolic blood pressure was measured by the tail pulse pick-up method, with rats in an unanesthetized condition. In experiment 1, five rats were used. The systolic blood pressure was measured once or twice a week. Urine volume and urinary protein were measured every day or every other day. Blood samples for the determination of PRC were collected before and 1 wk after the start of salt loading and thereafter twice a week in conscious rats from the tail. After recognition of stroke signs, such as repetitive lifting and convulsion of paws, paralysis of hindlimbs, or hyperirritability (33, 34), rats were killed and cerebral lesions were confirmed by autopsy. In experiments 2, 3, and 4, rats were maintained on 1% saline, and the systolic blood pressure and excretion of urinary protein were monitored as in experiment 1. Twoto-four weeks after saline loading, the rats were divided into three groups according to clinical findings such as proteinuria and stroke signs: group 1, rats without proteinuria; group 2, rats with proteinuria; and group 3, rats with both proteinuria and stroke signs. From the result of experiment 1, excretion of urinary protein greater than (100 mg/250 g body wt)/day was arbitrarily considered as positive proteinuria. Blood samples for PRC assay Society
H409
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were collected from the tail in conscious rats. Thus, in experiment 2, 25 rats were loaded with salt, and 6,8, and 11 rats were selected for groups 1, ZJand 3, respectively. Blood pressure responses to angiotensin II (AII) and norepinephrine (NE) were measured in these rats as well as in six age-matched rats on water. In experiment 3, from 15 stroke-prone SHR rats on 1% NaCl solution, 8 rats of group 1 and 7 rats of group 2 were obtained, and blood pressure response to an angiotensin II inhibitor was determined in these rats as well as in 7 age-matched rats on water. In experiment 4, from 12 rats on 1% saline, 4 rats of group 1, 4 rats of group 2, and 4 rats of group 3 were selected and used for histological examination. Blood pressure responses to angiotensin II and norepinephrine. Rats were anesthetized with urethan (750 mg/kg ip and 500 mg/kg SC),and polyethylene cannulas were placed in the femoral vein and external iliac artery. Mean arterial pressure was measured via the aortic catheter with a pressure transducer (MPU-0.5) and a polygraph (RM-45, Nihonkoden, Tokyo). Angiotensin II and NE were administered through the venous cannula in four doses ranging from 0.5 to 10 pg/kg and from 0.02 to 1 pg/kg, respectively. The log dose-response curve was assessedfor each agent in each rat and found to be linear up to 40 mmHg. The amount of pressor agent required to increase blood pressure by 30 mmHg was then calculated. Blood pressure responses to [l -sarcosine, 8-alanine]angiotensin II. The effects of an AII-inhibitor, [l-sarcosine, &alanine]AII, were determined in conscious rats. The femoral vein and external iliac artery were cannulated with polyethylene tubes (PE-10). These tubes were subcutaneously led to the back and exteriorized through a small skin puncture. Experiments were carried out at least 3 days after operation. After blood samples for PRC assay were collected from the tail blood vessels, the rats were placed in a transparent plastic cylinder (22 cm diam, 55 cm height). The drugs were injected through the venous catheter under unrestricted conditions. After an initial control period of about 30 min during which the blood pressure became stable, NE (0.5 pg/kg) and AI1 (0.05 pg/kg) were injected. Subsequently, the AIT inhib-
-
0
11 12 16 17 18 37 38
----
NAGAOKA,
SHINO,
AND
FUJITA
itor was infused for 40 min at the rate of 10 (pg/kg)/min because in rats that responded to the inhibitor, blood pressure reached the nadir 20-30 min after start of the infusion. Immediately and 40 min after the stop of the infusion, AI1 and NE were again injected to investigate the effect of the AI1 inhibitor. A representative recording for this assay is shown in Fig. 1. The effect of the inhibitor on blood pressure was manifested by the pressure difference before and 40 min after the infusion of the AI1 inhibitor. AnalyticaL methods. Urinary protein was determined by the biuret method (17). The amount of urinary protein was expressed in (mg/250 g)/day because most of the rats had a body weight of about 250 g. Plasma renin concentration was determined by radioimmunoassay for angiotensin I. Ten microliters of plasma were mixed with 40 ,ul of 2.5 ,uM rat angiotensinogen and 50 ~1 of 0.16 M phosphate buffer (pH 4.6) containing 0.35 mM dimercaprol and 0.174 mM &hydroxyquinoline. The mixture was incubated for 30 min at 37°C pH 5.7, and generated angiotensin I was measured by use of a radioimmunoassay kit (CEA-IRE-SORIN, France). Renin concentration is expressed in nanograms of angiotensin I generated in 1 h by 1 ml of plasma. Rat angiotensinogen was prepared according to the method of Haas et al. (20) from the plasma of Wistar-Kyoto rats nephrectomized 48 h previously. Statistical analysis was performed by Student’s t test for paired and unpaired comparisons and by linear regression analysis. Drugs. dl-Norepinephrine (Sankyo, Tokyo) and human angiotensin II (Protein Research Foundation, Osaka) were used. [ 1-Sarcosine, 8-alanine]AII was kindly supplied by Drs. Fujino and Fukuda (Chemical Research Laboratories) of our company. HistologicaL observations. The kidney and brain obtained from three groups of rats in experiment 4 were fixed in 10% neutral formaldehyde solution, embedded in paraffin, and cut into 4-pm sections. The sections were stained with hematoxylin-eosin and microscopically examined. All histological material was examined without knowledge of group. The intensity of histological changes
_-- --- -. .-
~
_____
FIG. 1. Effect of [I-sarcosine, &alaninelangiotensin II on mean blood pressure (MBP) in a rat with increased urinary protein. The angiotensin II inhibitor induced a fall in blood pressure and blocked the blood pressure response to
57 58 62 63 76 77 97 98 112
Time(mid Al hangiotensin
I I 50 nglkg,
NEvorepinephrine 500nglkg
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RENIN-ANGIOTENSIN Marks of individual Rot no. 1 Onset of stroke I I 0
IN
STROKE-PRONE
SHR
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rats 12 I I
I
IO
20
3 1
0
l
4
5
I I
c I
/
260 1
1
1
I
I
0
IO
20
30
tc
FIG. 2. Individual changes of systolic blood pressure (BP), plasma renin concentrations (PRC), excretion of urinary protein (UP), and urinary volume (UV) and onset of stroke in 5 stroke-prone SHR that were kept on 1% NaCl.
IL------L-----0
Days
on
I%
IO
-I--l--20
30
NaCl
was indicated by scale + to +++. Typical microscopic photographs of slight, moderate, and severe changes of hypertensive vascular lesions are shown in Fig. 6. RESULTS
Experiment 1. Blood pressure, PRC, urinary protein and volume, and stroke. Figure 2 demonstrates alterations in blood pressure, urinary protein, PRC, and urinary volume in five stroke-prone SHR from the start of 1% NaCl loading to the onset of stroke. The blood pressure was 193 t 7 mmHg (mean t SD) at the start of the experiment and progressively increased to about 230 mmHg, although the rate of blood pressure changes varied from rat to rat. After blood pressure attained 230 mmHg, excretion of urinary protein increased to levels higher than 100 (mg/250 g)/day. The time required for the onset of the higher urinary excretion of protein was 21 t 4.4 days for the five rats. Urinary volume also appeared to increase along with the elevation of urinary protein excretion. The stroke signs, such as repetitive lifting of forelimbs or head and hyperirritability, were observed 2-6 days after the prominent increase of urinary protein in each case. PRC increased along with elevation of urinary protein as shown in Fig. 2. For convenience, PRC values in Fig. 2 were rearranged on the basis of the clinical features of proteinuria and stroke signs (Fig. 3). PRC was 18.9 t 0.1 (ng/ml)/h at the start of experiment. After a week of salt loading, the value was suppressed to
z 0
100
a
50
ct
. -
Stroke
.
UP sign
Days on I%NaCl
-
-
o
7
-
-
18t5.1
2224.9
+
263.2
FIG. 3. Changes in plasma renin concentrations (PRC) of strokeprone SHR loaded with 1% NaCl solution. PRC data in Fig. 1 are rearranged as follows: before salt loading, 7 days after salt loading, about 3 days before increase in urinary protein (18 t 5.1 days), about 1 day after increase in urinary protein (22 t, 4.9 days), and at onset of stroke (26 -I- 5.2 days). Salt-loading duration required for increase in urinary protein was 21 t, 4.4 days for 5 rats. Values are means + SD. *P < 0.01; tP < 0.001 against rats on water.
10.9 t 3.1 (ng/ml)/h (P c 0.001). However, PRC gradually increased and reached 51.2 t 19.4 (ng/ml)/h (P < 0.01) at the stage of increased proteinuria. It further increased to 171 $- 88.6 (ng/ml)/h (P c 0.01) at the onset of stroke. Experiment 2. Pressor responses to AII and NE. Blood pressure responses to AI1 and NE are manifested
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TABLE 1. Blood pressure responses to norepinephrine and angiotensin II in stroke-prone SHR rats Criteria of Grouping Proteinuria
Rats
on water
Rats on 1% NaCl (days on 1% NaCl) Group 1 (21 -+ 10)
Stroke sign
No. of Rats
BP, mmHg
EDso AII, w/k
NE, P.gk
-
-
6
204 t17
2.2 k1.1
62 ~128
-
-
6
208 t8 228 t18 228 +14
1.6 to.7 1.4 to.6 1.6 to.8
22 t,12* 48 t40 75 t36
Group
2 (17 t, 8)
+
-
8
Group
3 (19 -+ 9)
+
+
11
Values are means t SD. EDso, dose of hormone producing 30 mmHg rise in blood pressure. Proteinuria + , L( 100 mg urinary protein/250 g)/ protein/250 g)/day. day. Proteinuria -, 0.05). Experiment 3. Effects of angiotensin blockade. The effect of [ 1-sarcosine, &alanine]AII on blood pressure was examined on stroke-prone SHR with different stages of hypertension (Table 2). The rats on tap water showed a slight pressor response to the AI1 inhibitor. One week after the start of NaCl loading, the rats without proteinuria (group 1) had no response to the inhibitor. However, rats with increased proteinuria (group 2) kept on 1% NaCl for about 2 wk showed a significant response to the AI1 inhibitor. A representative study in one of these rats is shown in Fig. 1. In these rats, the mean arterial pressure and PRC were increased. When individual response to the AI1 inhibitor was plotted against the amounts of PRC, there was a negative correlation between the response to the AI1 inhibitor and PRC (Fig. 5) . Experiment 4. Pathological observations. The results of histological observation are shown in Table 3. Rats were divided into three groups on the basis of clinical findings, as in experiment 2. Cerebral vascular lesions such as petechial hemorrhage and edema were found in rats with stroke signs ( group 3). Lesions were observed TABLE 2. Plasma renin concentration and effect of AII inhibitor on mean blood pressure of stroke-prone SHR
. I
50 100 150 PRC (ng/ml/hr) A UP 100 (mg/250 g)/day) and stroke signs: group 1, rats without increased proteinuria; group 2, rats with increased proteinuria; group 3, rats with both increased proteinuria and stroke signs. The response to NE did not change significantly among various experimental conditions. Sensitivity to AII, unlike the response to NE, was markedly influenced by the advance of malignant hypertension. When rats were kept on the NaCl solution, the ED30 decreased from 62 t 28 (mean t SD) to 22 t 12 rig/kg (P < 0.01). However, at the stage of appearance of proteinuria, the value tended to rise to 48 t 40 rig/kg, though not significantly (P > 0.05 vs. group I). In rats with stroke, the value was further increased to the level of 75 * 36 rig/kg (P < 0.01 vs. group l), which was comparable to that in rats on water (P > 0.05). When individual response to AI1 was plotted against PRC (Fig. 4), the sensitivity to AI1 was
t
r = 0.755 Plw&y
FIG. 5. Relation between plasma renin concentration change of mean blood pressure (MBP) following infusion sine, 8-alaninelangiotensin II. Rats were classified according water and amounts of urinary protein.
(PRC) and of [l-sarcoto drinking
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RENIN-ANGIOTENSIN
IN
STROKE-PRONE
SHR
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3. Histological changes in brain and kidney of stroke-prone SHR loaded with 1% NaCl
TABLE
Group
Rat Noa
1
2
Urinary Protein
Stroke Sign
PRC, (ng/mWh
1 2 3 4
-
-
9.5 10.5 11.7 11.9
-
-
5 6 7 8
+ + + +
-
20.0 70.8 75.0 148.0
-
-
++ ++ ++ ++
-
+
+ +++ ++ +++
++ +++ ++ ++
Urinary protein -, ((100 mg/250 g)/day; urinary protein +, ~(100 mg/250 g)/day. Other dashes indicate lack of stroke signs or histological changes. PRC, plasma renin concentration; T, thickening of arterial wall; FN, arterial fibrinoid necrosis; TA, tubular alterations such as hyaline cast and atrophy of tubules. Intensity of histological changes is indicat,ed by scale + to +++. *Hemorrhage was observed in the spinal cord.
in the cerebral cortex except in one rat (no. IO), which developed hemorrhage in the spinal cord. In the kidney, the interlobular arterial wall was focally and slightly thickened in rats without proteinuria (group 1 in Table 3, Fig. 6A). Thickening of vessel walls advanced in interlobular arteries and glomerular afferent arterioles in rats with increased proteinuria (group 2 in Table 3, Fig. 6B) and in rats with stroke (group 3 in Table 3, Fig. 6C). Fibrinoid necrosis of interlobular arteries was rarely observed in rats of group 1, but rats of groups 2 and 3 showed marked advanced lesions with arterial fibrinoid necrosis. Severity of fibrinoid necrosis appeared to be related to the elevation of PRC (Table 3). The glomeruli exhibited normal appearances in rats of group 1. In rats with increased proteinuria (groups 2 and 3), contraction and degeneration of capillary loops (Fig. 6, B and C), thickening and necrosis of intraglomerular arterioles (Fig. 6, B and C), and adhesion of the glomerular tuft to Bowman’s capsule were observed. Hyaline cast and atrophy of tubules were also remarkable in groups 2 and 3. From these findings, it appears that proteinuria and increase of PRC are related to renal vascular changes. DISCUSSION
In the present study, the development of severe hypertension and onset of stroke were induced in strokeprone SHR within 5 wk after loading 1% NaCl as drinking water, as reported by Nagaoka et al. (33) and Okamoto et al. (34). During the salt loading, increases in PRC and urinary protein and renovascular changes were confirmed between a steep rise of blood pressure up to 230 mmHg and the onset of stroke. Proteinuria, as well as neuroretinitis, is usually used as a clinical index for determination of severity of hypertension in humans (9, 14, 25, 35, 39). In addition, proteinuria
has been observed consistently in human malignant hypertension (24, 30). Thus, as in humans, an increase in urinary protein is likely to be one of the clinical features of malignant hypertension in these rats. Infusion of renin or AI1 was reported to induce proteinuria (1,38) through constriction of endothelial cells of glomerular capillaries (12). However, stroke-prone SHR with proteinuria showed vascular changes in glomeruli and tubular degeneration. This fact suggests proteinuria of these rats is more likely to be due to renal histological changes than to the elevation of renin or AII. In many cases of human malignant hypertension, plasma renin was reported to be increased (2, 5, 18, 19, 29), but in some cases to be suppressed (2, 22, 29). Similarly, the renin-angiotensin system was reported to be active in the cases of experimental malignant hypertension with Goldblatt hypertension (3, 26, 31, 32, 36) and spontaneous hypertension (28, 43), but was suppressed with DOCA-salt hypertension (15). From these facts, malignant vascular changes are not necessarily accompanied by activation of the renin-angiotensin system. However, the present studies indicated that elevation of PRC was well related to appearance of malignant vascular changes in the case of salt-loaded stroke-prone SHR rats. Thus, elevation of PRC as well as urinary protein is a convenient sign which indicates the onset of malignant hypertension in salt-loaded stroke-prone SHR rats. Because renin is an important determinant of AI1 generation, the elevation of plasma renin suggests an increase in the plasma level of AI1 in the rats in malignant phase. In the present study, the plasma level of AI1 in salt-loaded stroke-prone SHR was indirectly estimated by blood pressure response to AI1 as initially introduced by Kaplan and Silah (23). To avoid the development of tachyphylaxis to AI1 (42), the single bolus injection method was used in the present study. Usually, the response to AI1 has been indicated by the dose of AI1 producing 10 or 20 mmHg rise of blood pressure (8, 23). However, because log dose-response curves were found to be linear to 40 mmHg in stroke-prone SHR, the dose of AI1 producing 30 mmHg rise of blood pressure was used in this study (data not cited). By this method, the response to AI1 in salt-loaded stroke-prone SHR was found to decrease with elevation of PRC. This fact suggests the increase of endogenous AI1 level with the advance of hyperreninemia in salt-loaded stroke-prone SHR. The response to AI1 in salt-loaded rats with stroke was similar to that in rats on water. However, the endogenous level of AI1 appeared to be higher in rats with stroke than in rats on water, because the participation of the renin-angiotensin system in maintenance or elevation of hypertension was suggested by the hypotensive action of an AILinhibitor, [ I-sarcosine, 8-alanine]AII, in the malignant hypertensive phase of salt-loaded stroke-prone SHR as discussed below. The AI1 inhibitor [I-sarcosine, 8-alanine]AII (37) and its analogs have been used to indicate participation of the renin-angiotensin system in human renovascular or malignant hypertension (4, 7), in experimental renal hypertension (5, 6, 16, 21, 26, 27, 36), and in the malignant phase of stroke-prone SHR (43). In some of these studies,
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H414
SHIBOTA,
NAGAOKA,
SHINO, AND FUJITA
FIG. 6. Light microscopic pictures of the kidney from stroke-prone SHR rats on 1% NaCl solution. Hematoxylin-eosin stain, x130. A: kidney from a rat in group 1 (No. I). Focal thickening of an interlobular artery is observed. B: kidney from a rat in group 2 (No. 7j. Moderate thickening and fibrinoid necrosis of small arterioles are remarkable. Thickening and necrosis of glomerular arteries and contraction of capillary loops are observed in glomeruli. Hyaline cast formation and tubular atrophy are also remarkably observed. C: kidney from a rat in group 3 (No. 11). Fibrinoid necrosis and proliferative arteriosclerosis with hemorrhage are remarkably observed. Tubular atrophy and glomerular degeneration are also remarkably observed.
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RENIN-ANGIOTENSIN
IN
STROKE-PRONE
SHR
H415
RATS
a negative correlation was observed between PRC and the blood pressure change due to the inhibitor (7, 26, 27, 36, 43). A similar negative correlation was obtained in the present study with salt-loaded stroke-prone SHR with malignant phase. Therefore the renin-angiotensin system seems to be active in the malignant phase of hypertension in salt-loaded stroke-prone SHR. Mohring et al. (31, 32) postulated that in the late stage of two-kidney Goldblatt hypertensive rats, pressure diuresis and natriuresis induced an increase of plasma renin activity that maintained or further increased blood pressure level. The high blood pressure levels and high renin activities are presumed to induce vascular damage and deterioration of renal function. In the present study, development of vascular lesions in the kidney and brain was observed with increase in PRC in the malignant phase of sah-loaded stroke-prone SHR. Therefore, the postulation of Mohring et al. in Goldblatt hypertension may also be valid in the malignant phase of salt-loaded stroke-prone SHR, although the occurrence of natriuresis and hypovolemia was not examined in the present study. However, an increase in urinary volume did not precede increases in PRC and urinary excretion of protein. Among the rats with increased urinary protein, the PRC level appeared to be parallel with renovascular changes. Together with these findings, it is postulated that renal vascular changes due to severe hypertension result in the
activat,ion of renin secretion through reduction of renal perfusion pressure. Work from other laboratories showed that plasma renin levels were unchanged (l-0,13) or decreased (11,40, 41) with the advance of hypertension in regular SHR that hardly developed malignant hypertension. In contrast, in the malignant hypertensive phase of salt-loaded stroke-prone SHR rats, the act!ivated renin-angiotensin system appears to maintain hypertension. This activation of the renin-angiotensin system in the salt-loaded condition is similar to that in the malignant phase of spontaneous hypertension of stroke-prone SHR as reported by Matsunaga et al. (28) and Yamamoto (43). According to their reports, hyperreninemia was established in stroke-prone SHR at 5-8 mo of age when they had cerebra- or renal vascular lesions. The infusion of an AII-inhibitor, [1-sarcosine, &isoleucine]AII, caused a significant fall in blood pressure of the stroke-prone SHR with hyperreninemia. Thus, salt loading appears to be a useful method to reduce the period required for the development of malignant hypertension in stroke-prone SHR. ’ The authors the preparation Received
14 Nov
thank Dr. H. Iwat.suka of this manuscript. 1977; accepted
in final
for his valuable form
24 Ott
discussion
in
1978.
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pressure and plasma renin activity in the spontaneously hypertensive rat. Circ. Res. 37: 66-71, 1975. FREIS, E. D. A double blind control study of antihypertensive agents. I. Comparative effectiveness of reserpine, reserpine and hydralazine, and three ganglion blocking agents, chlorisodamine, mecamylamine and pentolinium tartrate. Arch. Intern. Med. 106: 81-96, 1960. GAVRAS, H., H. R. BRUNNER, J. H. LARAGH, E. D. VAUGHAN, JR.., M. Koss, L. J. COTE, AND I. GAVRAS. Malignant hypertension resulting from deoxycorticosterone acetate and salt excess. Circ. Res, 36: 300-309, 1975. GAVRAS, H., H. R. BRUNNER, E. D. VAUGHAN, JR., AND J. H. LARAGH, Angiotensin-sodium interaction in blcod pressure maintenance of renal hypertension and normotensive rats. Science 180: 1369-1372, 1973. GORNELL, A, G., C. J. BARDAWILL, AND M. M. DAVID. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: ‘751-766, 1949. GOULD, A. B., L. T. &EGGS, AND J. R. KSHAN. Measurement of renin and substrate concentrations in human serum. Lab, Incest. 15: 1802-1813, 1966. GUNNELLS, 9. C., JR., C. E. GRIM, R. R,. ROBINSON, AND N. M. WILI~ERMANN. Plasma renin activity in healthy subjects and patients with hypertension. Arch. Intern. Med. 119: 232-240, 1967. HAAS, E., H. GOLDBLATT, E. C. GIPSON, AND L. LEWIS. Extraction, purification, and assay of human renin free of angiotensinase. Circ. Res. 19: 739-749, 1966. JOHNSON, J. A., J. 0. DAVIS, AND B. BRAVERMAN. Role of angiotensin II in experimental renal hypertension in the rabbit. Am. ?J. Physiol. 228: 11-16, 1975. KAPLAN, N. M. Primary aldosteronism with malignant hypertension. IV. Engl. J. Med. 269: 1282-1286, 1963. KAPI,AN, N. M., AND J. G. SILAH. The angiotensin infusion test: a new approach to the differential diagnosis of renovascular hypertension. N. Engl. J. Med. 271: 536-551, 1964. KINCAID-SMITH, P., J. MCMICHAEL, AND E. A. MURPHY. The clinical course and pathology of hypertension with papilloedema (malignant hypertension). Q. J. Med. 27: 117-153, 1958. KIRKENDALL, W. M., AND J. W. CULBERTSON. Managernent of
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H416 systemic 1955.
SHIBOTA, arterial
hypertension.
Arch.
Intern.
Med.
95: 601-613,
26. LOHMEIER, T. E., AND J. 0. DAVIS. Renin-angiotensin-aldosterone system in experimental renal hypertension in the rabbit. Am. J. Physiol. 230: 311-318, 1976. 27. MACDONALD, G. J., G. W. BOYD, AND W. S. PEART. Effect of the angiotensin II blocker l-sar-&ala-angiotensin II on renal artery clip hypertension in the rat. Circ. Res. 37: 640-646, 1975. 28. MATSUNAGA, M., J. YAMAMOTO, A. HARA, Y. YAMORI, K. OGINO, AND K. OKAMOTO. Plasma renin and hypertensive vascular complications: an observation in the stroke-prone spontaneously hypertensive rat. Jpn. Circ. J. 39: 1305-1311, 1975. 29. MCALLISTER, R. C., JR., C. W. VAN WAY III, K. DAYANI, W. J. ANDERSON, E. TEMPLES, A. M. MICHELAKIS, W. S. COPPAGE, AND J. A. OATES. Malignant hypertension: effects of therapy on renin and aldosterone. Circ. Res. 28, Suppl. 2: 160-174, 1971. 30. MILLIEZ, P., P. TCHERDAKOFF, P. SAMRCQ, AND L. P. REY. In: Essential hypertension, edited by K. D. Bock and P. T. Cottier. Berlin-Gottingen-Heidelberg: Springer-Verlag, 1960, p. 214-230. 31. M~HRING, J., B. M~HRING, H.-J., N&MANN, A. PHILIPPI, E. HOMSY, H. ORTH, G. DAUDA, S. KAZDA, AND F. GROSS. Salt and water balance and renin activity in renal hypertension of rats. Am. J. Physiol. 228: 1847-1855, 1975. 32. M~HRING, J., M. PETRI, M. SZOKOL, D. HAACK, AND B. M~HRING. Effects of saline drinking on malignant course of renal hypertension in rats. Am. J. Physiol. 230: 849-857, 1976. 33. NAGAOKA, A., H. IWATSUKA, 2. SUZUOKI, AND K. OKAMOTO. Genetic predisposition to stroke in spontaneously hypertensive rats.
NAGAOKA,
SHINO,
AND
FUJITA
Am. J. PhysioZ. 230: 1354-1359, 1976. 34. OKAMOTO, K., Y. YAMORI, AND A. NAGAOKA. Establishment of the stroke-prone spontaneously hypertensive rat (SHR). Circ. Res. 3435, suppz. 1: 143-153, 1974. 35. PALMER, R. S., D. LOOFBOUROW, AND C. R. DOERING. Progress in essential hypertension. Eight-year follow-up study of 430 patients on conventional medical treatment. N. EngZ. J. Med. 239: 990-994, 1948. 36. PALS, D. T., AND F. D. MASUCCI. Plasma renin and the antihypertensive effect of 1-sar-8-ala-angiotensin II. Eur. J. PharmacoZ. 23: 115-119, 1973. 37. PALS, D. T., F. D. MASUCCI, G. S. DENNING, F. SIPAS, AND D. C. FESSLER. Role of the pressor action of angiotensin II in experimental hypertension. Circ. Res. 29: 673-681, 1971. 38. PICKERING, G. W., AND M. PRINZMETAL. The effect of renin on urine formation. J. PhysioZ London 98: 314-335, 1940. 39. SCHROEDER, H. A. Management of arterial hypertension. Am. J. Med. 17: 540-561, 1954. 40. SEN, S., R. R. SMEBY, AND F. M. BUMPUS. Renin in rats with spontaneous hypertension. Circ. Res. 31: 876-880, 1972. 41. SHIONO K., AND H. SOKABE. Renin-angiotensin system in spontaneously hypertensive rats. Am. J. PhysioZ. 231: 1295-1299, 1976. 42. SLACK, B., AND J. M. LEDINGHAM. The influence of sodium intake on the pressor response to angiotensin II in the unanesthetized rat. CZin. Sci. MOL. Med. 50: 285-291, 1976. 43. YAMAMOTO, J. Role of renin-angiotensin system in experimental hypertension in rats: plasma renin and hypotensive effect of angiotensin II antagonist. Jpn. Circ. J. 41: 665-675, 1977.
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SHIBOTA, MASAKI, AKINOBU NAGAOKA, AKIO SHINO, AND TAKESHI FUJITA. Renin-angiotensin system in stroke-prone spontaneously hypertensive rats. Am. J. Physiol. 236(3): H409H416, 1979 or Am. J. Physiol: Heart Circ. Physiol. 5(3): H409H416, 1979.-The development of malignant hypertension was studied in stroke-prone spontaneously hypertensive rats (SHR) kept on 1% NaCl as drinking water. Along with salt-loading, blood pressure gradually increased and reached a severe hypertensive level (>230 mmHg), which was followed by increases in urinary protein (X00 (mg/250 g body wt)/day) and plasma renin concentration (PRC, from 18.9 t 0.1 to 51.2 & 19.4 (ng/ ml)/h, mean t SD). At this stage, renal small arteries and arterioles showed severe sclerosis and fibrinoid necrosis. Stroke was observed within a week after the onset of these renal abnormalities. The dose of exogenous angiotensin II (AII) producing 30 mmHg rise in blood pressure increased with the elevation of PRC, from 22 t, 12 to 75 t 36 rig/kg, which was comparable to that in rats on water. The fall of blood pressure due to an AI1 inhibitor, [1-sarcosine, &alanine]AII (10 (pg/kg)/ min for 40 min) became more prominent with the increase in PRC in salt-loaded rats, but was not detected in rats on water. These findings suggest that the activation of renin-angiotensin system participates in malignant hypertension of salt-loaded stroke-prone SHR rats that show stroke signs, proteinuria, hyperreninemia, and renovascular changes. salt-loading; urinary lignant hypertension; inhibitor
protein; plasma renin concentration; maresponse to angiotensin II; angiotensin II
SPONTANEOUSLY HYPERTENSIVE RATS (SHR)have been used as a model of human hypertension. Recently, Okamoto et al. (34) established a substrain (stroke-prone SHR rats) from the SHR rats; these rats were more hypertensive and developed cerebrovascular lesions (CVL) with a higher incidence than the original strain. Both the development of hypertension and onset of CVL of stroke-prone SHR rats were accelerated by salt loading (33,34). When the rats were kept on 1% NaCl as drinking water at the age of about 10 wk, most of them showed severe hypertension (>220 mmHg) and developed CVL within 5 wk. These reports suggested that the salt-loaded stroke-prone SHR is an excellent model for studies of malignant hypertension. Proteinuria is usually used as a clinical index in human hypertension (9, 14,25,35, 39). In the malignant phase of hypertension, proteinuria (24, 30) and hyperreninemia 0363-6135/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
(2, 5, 18, 19, 29) are often observed, although some investigators reported some casesof malignant hypertension without hyperreninemia (2, 22, 29). Similarly in animals, the activation of the renin-angiotensin system was reported in the malignant phase of experimental renovascular (3, 26, 31, 32, 36) or spontaneous (28, 43) hypertension. The present study was designed to correlate changes in excretion of urinary protein and plasma renin concentration (PRC) with changes in blood pressure and malignant vascular changes in stroke-prone SHR during prolonged administration of 1% NaCl solution. Roles of the renin-angiotensin system were also examined in the malignant phase hypertension. METHODS
Animals. Experiments were performed on male strokeprone SHR. They were maintained on a laboratory chow, CA-l, containing 10.4 meq of sodium/l00 g (Japan Clea, Tokyo) and water ad libitum. Salt loading was started from about 11 wk of age by giving 1% NaCl as drinking water. Individual metabolic cageswere used for collection of urine. The systolic blood pressure was measured by the tail pulse pick-up method, with rats in an unanesthetized condition. In experiment 1, five rats were used. The systolic blood pressure was measured once or twice a week. Urine volume and urinary protein were measured every day or every other day. Blood samples for the determination of PRC were collected before and 1 wk after the start of salt loading and thereafter twice a week in conscious rats from the tail. After recognition of stroke signs, such as repetitive lifting and convulsion of paws, paralysis of hindlimbs, or hyperirritability (33, 34), rats were killed and cerebral lesions were confirmed by autopsy. In experiments 2, 3, and 4, rats were maintained on 1% saline, and the systolic blood pressure and excretion of urinary protein were monitored as in experiment 1. Twoto-four weeks after saline loading, the rats were divided into three groups according to clinical findings such as proteinuria and stroke signs: group 1, rats without proteinuria; group 2, rats with proteinuria; and group 3, rats with both proteinuria and stroke signs. From the result of experiment 1, excretion of urinary protein greater than (100 mg/250 g body wt)/day was arbitrarily considered as positive proteinuria. Blood samples for PRC assay Society
H409
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H410
SHIBOTA,
were collected from the tail in conscious rats. Thus, in experiment 2, 25 rats were loaded with salt, and 6,8, and 11 rats were selected for groups 1, ZJand 3, respectively. Blood pressure responses to angiotensin II (AII) and norepinephrine (NE) were measured in these rats as well as in six age-matched rats on water. In experiment 3, from 15 stroke-prone SHR rats on 1% NaCl solution, 8 rats of group 1 and 7 rats of group 2 were obtained, and blood pressure response to an angiotensin II inhibitor was determined in these rats as well as in 7 age-matched rats on water. In experiment 4, from 12 rats on 1% saline, 4 rats of group 1, 4 rats of group 2, and 4 rats of group 3 were selected and used for histological examination. Blood pressure responses to angiotensin II and norepinephrine. Rats were anesthetized with urethan (750 mg/kg ip and 500 mg/kg SC),and polyethylene cannulas were placed in the femoral vein and external iliac artery. Mean arterial pressure was measured via the aortic catheter with a pressure transducer (MPU-0.5) and a polygraph (RM-45, Nihonkoden, Tokyo). Angiotensin II and NE were administered through the venous cannula in four doses ranging from 0.5 to 10 pg/kg and from 0.02 to 1 pg/kg, respectively. The log dose-response curve was assessedfor each agent in each rat and found to be linear up to 40 mmHg. The amount of pressor agent required to increase blood pressure by 30 mmHg was then calculated. Blood pressure responses to [l -sarcosine, 8-alanine]angiotensin II. The effects of an AII-inhibitor, [l-sarcosine, &alanine]AII, were determined in conscious rats. The femoral vein and external iliac artery were cannulated with polyethylene tubes (PE-10). These tubes were subcutaneously led to the back and exteriorized through a small skin puncture. Experiments were carried out at least 3 days after operation. After blood samples for PRC assay were collected from the tail blood vessels, the rats were placed in a transparent plastic cylinder (22 cm diam, 55 cm height). The drugs were injected through the venous catheter under unrestricted conditions. After an initial control period of about 30 min during which the blood pressure became stable, NE (0.5 pg/kg) and AI1 (0.05 pg/kg) were injected. Subsequently, the AIT inhib-
-
0
11 12 16 17 18 37 38
----
NAGAOKA,
SHINO,
AND
FUJITA
itor was infused for 40 min at the rate of 10 (pg/kg)/min because in rats that responded to the inhibitor, blood pressure reached the nadir 20-30 min after start of the infusion. Immediately and 40 min after the stop of the infusion, AI1 and NE were again injected to investigate the effect of the AI1 inhibitor. A representative recording for this assay is shown in Fig. 1. The effect of the inhibitor on blood pressure was manifested by the pressure difference before and 40 min after the infusion of the AI1 inhibitor. AnalyticaL methods. Urinary protein was determined by the biuret method (17). The amount of urinary protein was expressed in (mg/250 g)/day because most of the rats had a body weight of about 250 g. Plasma renin concentration was determined by radioimmunoassay for angiotensin I. Ten microliters of plasma were mixed with 40 ,ul of 2.5 ,uM rat angiotensinogen and 50 ~1 of 0.16 M phosphate buffer (pH 4.6) containing 0.35 mM dimercaprol and 0.174 mM &hydroxyquinoline. The mixture was incubated for 30 min at 37°C pH 5.7, and generated angiotensin I was measured by use of a radioimmunoassay kit (CEA-IRE-SORIN, France). Renin concentration is expressed in nanograms of angiotensin I generated in 1 h by 1 ml of plasma. Rat angiotensinogen was prepared according to the method of Haas et al. (20) from the plasma of Wistar-Kyoto rats nephrectomized 48 h previously. Statistical analysis was performed by Student’s t test for paired and unpaired comparisons and by linear regression analysis. Drugs. dl-Norepinephrine (Sankyo, Tokyo) and human angiotensin II (Protein Research Foundation, Osaka) were used. [ 1-Sarcosine, 8-alanine]AII was kindly supplied by Drs. Fujino and Fukuda (Chemical Research Laboratories) of our company. HistologicaL observations. The kidney and brain obtained from three groups of rats in experiment 4 were fixed in 10% neutral formaldehyde solution, embedded in paraffin, and cut into 4-pm sections. The sections were stained with hematoxylin-eosin and microscopically examined. All histological material was examined without knowledge of group. The intensity of histological changes
_-- --- -. .-
~
_____
FIG. 1. Effect of [I-sarcosine, &alaninelangiotensin II on mean blood pressure (MBP) in a rat with increased urinary protein. The angiotensin II inhibitor induced a fall in blood pressure and blocked the blood pressure response to
57 58 62 63 76 77 97 98 112
Time(mid Al hangiotensin
I I 50 nglkg,
NEvorepinephrine 500nglkg
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RENIN-ANGIOTENSIN Marks of individual Rot no. 1 Onset of stroke I I 0
IN
STROKE-PRONE
SHR
H411
RATS
rats 12 I I
I
IO
20
3 1
0
l
4
5
I I
c I
/
260 1
1
1
I
I
0
IO
20
30
tc
FIG. 2. Individual changes of systolic blood pressure (BP), plasma renin concentrations (PRC), excretion of urinary protein (UP), and urinary volume (UV) and onset of stroke in 5 stroke-prone SHR that were kept on 1% NaCl.
IL------L-----0
Days
on
I%
IO
-I--l--20
30
NaCl
was indicated by scale + to +++. Typical microscopic photographs of slight, moderate, and severe changes of hypertensive vascular lesions are shown in Fig. 6. RESULTS
Experiment 1. Blood pressure, PRC, urinary protein and volume, and stroke. Figure 2 demonstrates alterations in blood pressure, urinary protein, PRC, and urinary volume in five stroke-prone SHR from the start of 1% NaCl loading to the onset of stroke. The blood pressure was 193 t 7 mmHg (mean t SD) at the start of the experiment and progressively increased to about 230 mmHg, although the rate of blood pressure changes varied from rat to rat. After blood pressure attained 230 mmHg, excretion of urinary protein increased to levels higher than 100 (mg/250 g)/day. The time required for the onset of the higher urinary excretion of protein was 21 t 4.4 days for the five rats. Urinary volume also appeared to increase along with the elevation of urinary protein excretion. The stroke signs, such as repetitive lifting of forelimbs or head and hyperirritability, were observed 2-6 days after the prominent increase of urinary protein in each case. PRC increased along with elevation of urinary protein as shown in Fig. 2. For convenience, PRC values in Fig. 2 were rearranged on the basis of the clinical features of proteinuria and stroke signs (Fig. 3). PRC was 18.9 t 0.1 (ng/ml)/h at the start of experiment. After a week of salt loading, the value was suppressed to
z 0
100
a
50
ct
. -
Stroke
.
UP sign
Days on I%NaCl
-
-
o
7
-
-
18t5.1
2224.9
+
263.2
FIG. 3. Changes in plasma renin concentrations (PRC) of strokeprone SHR loaded with 1% NaCl solution. PRC data in Fig. 1 are rearranged as follows: before salt loading, 7 days after salt loading, about 3 days before increase in urinary protein (18 t 5.1 days), about 1 day after increase in urinary protein (22 t, 4.9 days), and at onset of stroke (26 -I- 5.2 days). Salt-loading duration required for increase in urinary protein was 21 t, 4.4 days for 5 rats. Values are means + SD. *P < 0.01; tP < 0.001 against rats on water.
10.9 t 3.1 (ng/ml)/h (P c 0.001). However, PRC gradually increased and reached 51.2 t 19.4 (ng/ml)/h (P < 0.01) at the stage of increased proteinuria. It further increased to 171 $- 88.6 (ng/ml)/h (P c 0.01) at the onset of stroke. Experiment 2. Pressor responses to AII and NE. Blood pressure responses to AI1 and NE are manifested
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H412
SHIBOTA,
TABLE 1. Blood pressure responses to norepinephrine and angiotensin II in stroke-prone SHR rats Criteria of Grouping Proteinuria
Rats
on water
Rats on 1% NaCl (days on 1% NaCl) Group 1 (21 -+ 10)
Stroke sign
No. of Rats
BP, mmHg
EDso AII, w/k
NE, P.gk
-
-
6
204 t17
2.2 k1.1
62 ~128
-
-
6
208 t8 228 t18 228 +14
1.6 to.7 1.4 to.6 1.6 to.8
22 t,12* 48 t40 75 t36
Group
2 (17 t, 8)
+
-
8
Group
3 (19 -+ 9)
+
+
11
Values are means t SD. EDso, dose of hormone producing 30 mmHg rise in blood pressure. Proteinuria + , L( 100 mg urinary protein/250 g)/ protein/250 g)/day. day. Proteinuria -, 0.05). Experiment 3. Effects of angiotensin blockade. The effect of [ 1-sarcosine, &alanine]AII on blood pressure was examined on stroke-prone SHR with different stages of hypertension (Table 2). The rats on tap water showed a slight pressor response to the AI1 inhibitor. One week after the start of NaCl loading, the rats without proteinuria (group 1) had no response to the inhibitor. However, rats with increased proteinuria (group 2) kept on 1% NaCl for about 2 wk showed a significant response to the AI1 inhibitor. A representative study in one of these rats is shown in Fig. 1. In these rats, the mean arterial pressure and PRC were increased. When individual response to the AI1 inhibitor was plotted against the amounts of PRC, there was a negative correlation between the response to the AI1 inhibitor and PRC (Fig. 5) . Experiment 4. Pathological observations. The results of histological observation are shown in Table 3. Rats were divided into three groups on the basis of clinical findings, as in experiment 2. Cerebral vascular lesions such as petechial hemorrhage and edema were found in rats with stroke signs ( group 3). Lesions were observed TABLE 2. Plasma renin concentration and effect of AII inhibitor on mean blood pressure of stroke-prone SHR
. I
50 100 150 PRC (ng/ml/hr) A UP 100 (mg/250 g)/day) and stroke signs: group 1, rats without increased proteinuria; group 2, rats with increased proteinuria; group 3, rats with both increased proteinuria and stroke signs. The response to NE did not change significantly among various experimental conditions. Sensitivity to AII, unlike the response to NE, was markedly influenced by the advance of malignant hypertension. When rats were kept on the NaCl solution, the ED30 decreased from 62 t 28 (mean t SD) to 22 t 12 rig/kg (P < 0.01). However, at the stage of appearance of proteinuria, the value tended to rise to 48 t 40 rig/kg, though not significantly (P > 0.05 vs. group I). In rats with stroke, the value was further increased to the level of 75 * 36 rig/kg (P < 0.01 vs. group l), which was comparable to that in rats on water (P > 0.05). When individual response to AI1 was plotted against PRC (Fig. 4), the sensitivity to AI1 was
t
r = 0.755 Plw&y
FIG. 5. Relation between plasma renin concentration change of mean blood pressure (MBP) following infusion sine, 8-alaninelangiotensin II. Rats were classified according water and amounts of urinary protein.
(PRC) and of [l-sarcoto drinking
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RENIN-ANGIOTENSIN
IN
STROKE-PRONE
SHR
H413
RATS
3. Histological changes in brain and kidney of stroke-prone SHR loaded with 1% NaCl
TABLE
Group
Rat Noa
1
2
Urinary Protein
Stroke Sign
PRC, (ng/mWh
1 2 3 4
-
-
9.5 10.5 11.7 11.9
-
-
5 6 7 8
+ + + +
-
20.0 70.8 75.0 148.0
-
-
++ ++ ++ ++
-
+
+ +++ ++ +++
++ +++ ++ ++
Urinary protein -, ((100 mg/250 g)/day; urinary protein +, ~(100 mg/250 g)/day. Other dashes indicate lack of stroke signs or histological changes. PRC, plasma renin concentration; T, thickening of arterial wall; FN, arterial fibrinoid necrosis; TA, tubular alterations such as hyaline cast and atrophy of tubules. Intensity of histological changes is indicat,ed by scale + to +++. *Hemorrhage was observed in the spinal cord.
in the cerebral cortex except in one rat (no. IO), which developed hemorrhage in the spinal cord. In the kidney, the interlobular arterial wall was focally and slightly thickened in rats without proteinuria (group 1 in Table 3, Fig. 6A). Thickening of vessel walls advanced in interlobular arteries and glomerular afferent arterioles in rats with increased proteinuria (group 2 in Table 3, Fig. 6B) and in rats with stroke (group 3 in Table 3, Fig. 6C). Fibrinoid necrosis of interlobular arteries was rarely observed in rats of group 1, but rats of groups 2 and 3 showed marked advanced lesions with arterial fibrinoid necrosis. Severity of fibrinoid necrosis appeared to be related to the elevation of PRC (Table 3). The glomeruli exhibited normal appearances in rats of group 1. In rats with increased proteinuria (groups 2 and 3), contraction and degeneration of capillary loops (Fig. 6, B and C), thickening and necrosis of intraglomerular arterioles (Fig. 6, B and C), and adhesion of the glomerular tuft to Bowman’s capsule were observed. Hyaline cast and atrophy of tubules were also remarkable in groups 2 and 3. From these findings, it appears that proteinuria and increase of PRC are related to renal vascular changes. DISCUSSION
In the present study, the development of severe hypertension and onset of stroke were induced in strokeprone SHR within 5 wk after loading 1% NaCl as drinking water, as reported by Nagaoka et al. (33) and Okamoto et al. (34). During the salt loading, increases in PRC and urinary protein and renovascular changes were confirmed between a steep rise of blood pressure up to 230 mmHg and the onset of stroke. Proteinuria, as well as neuroretinitis, is usually used as a clinical index for determination of severity of hypertension in humans (9, 14, 25, 35, 39). In addition, proteinuria
has been observed consistently in human malignant hypertension (24, 30). Thus, as in humans, an increase in urinary protein is likely to be one of the clinical features of malignant hypertension in these rats. Infusion of renin or AI1 was reported to induce proteinuria (1,38) through constriction of endothelial cells of glomerular capillaries (12). However, stroke-prone SHR with proteinuria showed vascular changes in glomeruli and tubular degeneration. This fact suggests proteinuria of these rats is more likely to be due to renal histological changes than to the elevation of renin or AII. In many cases of human malignant hypertension, plasma renin was reported to be increased (2, 5, 18, 19, 29), but in some cases to be suppressed (2, 22, 29). Similarly, the renin-angiotensin system was reported to be active in the cases of experimental malignant hypertension with Goldblatt hypertension (3, 26, 31, 32, 36) and spontaneous hypertension (28, 43), but was suppressed with DOCA-salt hypertension (15). From these facts, malignant vascular changes are not necessarily accompanied by activation of the renin-angiotensin system. However, the present studies indicated that elevation of PRC was well related to appearance of malignant vascular changes in the case of salt-loaded stroke-prone SHR rats. Thus, elevation of PRC as well as urinary protein is a convenient sign which indicates the onset of malignant hypertension in salt-loaded stroke-prone SHR rats. Because renin is an important determinant of AI1 generation, the elevation of plasma renin suggests an increase in the plasma level of AI1 in the rats in malignant phase. In the present study, the plasma level of AI1 in salt-loaded stroke-prone SHR was indirectly estimated by blood pressure response to AI1 as initially introduced by Kaplan and Silah (23). To avoid the development of tachyphylaxis to AI1 (42), the single bolus injection method was used in the present study. Usually, the response to AI1 has been indicated by the dose of AI1 producing 10 or 20 mmHg rise of blood pressure (8, 23). However, because log dose-response curves were found to be linear to 40 mmHg in stroke-prone SHR, the dose of AI1 producing 30 mmHg rise of blood pressure was used in this study (data not cited). By this method, the response to AI1 in salt-loaded stroke-prone SHR was found to decrease with elevation of PRC. This fact suggests the increase of endogenous AI1 level with the advance of hyperreninemia in salt-loaded stroke-prone SHR. The response to AI1 in salt-loaded rats with stroke was similar to that in rats on water. However, the endogenous level of AI1 appeared to be higher in rats with stroke than in rats on water, because the participation of the renin-angiotensin system in maintenance or elevation of hypertension was suggested by the hypotensive action of an AILinhibitor, [ I-sarcosine, 8-alanine]AII, in the malignant hypertensive phase of salt-loaded stroke-prone SHR as discussed below. The AI1 inhibitor [I-sarcosine, 8-alanine]AII (37) and its analogs have been used to indicate participation of the renin-angiotensin system in human renovascular or malignant hypertension (4, 7), in experimental renal hypertension (5, 6, 16, 21, 26, 27, 36), and in the malignant phase of stroke-prone SHR (43). In some of these studies,
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H414
SHIBOTA,
NAGAOKA,
SHINO, AND FUJITA
FIG. 6. Light microscopic pictures of the kidney from stroke-prone SHR rats on 1% NaCl solution. Hematoxylin-eosin stain, x130. A: kidney from a rat in group 1 (No. I). Focal thickening of an interlobular artery is observed. B: kidney from a rat in group 2 (No. 7j. Moderate thickening and fibrinoid necrosis of small arterioles are remarkable. Thickening and necrosis of glomerular arteries and contraction of capillary loops are observed in glomeruli. Hyaline cast formation and tubular atrophy are also remarkably observed. C: kidney from a rat in group 3 (No. 11). Fibrinoid necrosis and proliferative arteriosclerosis with hemorrhage are remarkably observed. Tubular atrophy and glomerular degeneration are also remarkably observed.
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RENIN-ANGIOTENSIN
IN
STROKE-PRONE
SHR
H415
RATS
a negative correlation was observed between PRC and the blood pressure change due to the inhibitor (7, 26, 27, 36, 43). A similar negative correlation was obtained in the present study with salt-loaded stroke-prone SHR with malignant phase. Therefore the renin-angiotensin system seems to be active in the malignant phase of hypertension in salt-loaded stroke-prone SHR. Mohring et al. (31, 32) postulated that in the late stage of two-kidney Goldblatt hypertensive rats, pressure diuresis and natriuresis induced an increase of plasma renin activity that maintained or further increased blood pressure level. The high blood pressure levels and high renin activities are presumed to induce vascular damage and deterioration of renal function. In the present study, development of vascular lesions in the kidney and brain was observed with increase in PRC in the malignant phase of sah-loaded stroke-prone SHR. Therefore, the postulation of Mohring et al. in Goldblatt hypertension may also be valid in the malignant phase of salt-loaded stroke-prone SHR, although the occurrence of natriuresis and hypovolemia was not examined in the present study. However, an increase in urinary volume did not precede increases in PRC and urinary excretion of protein. Among the rats with increased urinary protein, the PRC level appeared to be parallel with renovascular changes. Together with these findings, it is postulated that renal vascular changes due to severe hypertension result in the
activat,ion of renin secretion through reduction of renal perfusion pressure. Work from other laboratories showed that plasma renin levels were unchanged (l-0,13) or decreased (11,40, 41) with the advance of hypertension in regular SHR that hardly developed malignant hypertension. In contrast, in the malignant hypertensive phase of salt-loaded stroke-prone SHR rats, the act!ivated renin-angiotensin system appears to maintain hypertension. This activation of the renin-angiotensin system in the salt-loaded condition is similar to that in the malignant phase of spontaneous hypertension of stroke-prone SHR as reported by Matsunaga et al. (28) and Yamamoto (43). According to their reports, hyperreninemia was established in stroke-prone SHR at 5-8 mo of age when they had cerebra- or renal vascular lesions. The infusion of an AII-inhibitor, [1-sarcosine, &isoleucine]AII, caused a significant fall in blood pressure of the stroke-prone SHR with hyperreninemia. Thus, salt loading appears to be a useful method to reduce the period required for the development of malignant hypertension in stroke-prone SHR. ’ The authors the preparation Received
14 Nov
thank Dr. H. Iwat.suka of this manuscript. 1977; accepted
in final
for his valuable form
24 Ott
discussion
in
1978.
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