AJH
1992;5:671-680
ORIGINAL CONTRIBUTIONS
S. Chen, C. Yuan, J.F. Schooley, Jr., F.J. Haddy, and M.B. Pamnani
Hypertension is frequently seen in insulindependent diabetes mellitus (IDDM), but the mechanism of the hypertension is unknown. An animal model of IDDM hypertension could be helpful in determining the mechanism, but experimental IDDM has been infrequently and irregularly associated with hypertension. In an attempt to develop a consistent model of IDDM hypertension, we superimposed streptozotocin (STZ)-induced IDDM on surgical reduction of renal mass (RRM) in Wistar rats. Seven groups of rats were studied: 1) 60% RRM receiving 65 mg/kg body weight (BW) STZ; 2) 60% RRM receiving 40 mg/kg BW STZ; 3) 25% RRM receiving 65 mg/kg BW STZ; 4) two kidney normal rats receiving 65 mg/kg BW STZ; 5) 60% RRM receiving vehicle (control for group 1); 6) 60% RRM receiving vehicle (control for group 2); and 7) 25% RRM receiving vehicle. STZ produced diabetes and hypertension within 1 to 2 weeks in all three group's of RRM rats but blood pressure was unaffected by 60% or 25% RRM alone. STZ alone had no
H
ypertension is frequently seen in insulindependent diabetes mellitus (IDDM). " The mechanism of this hypertension is unknown. An animal model of IDDM hypertension might be useful in probing the mechanism of this type of hypertension but attempts to develop such a consistent 1
3
effect on blood pressure until the 5th week when the blood pressure increased slightly. Progressive weight loss resulted from 65 mg/kg BW STZ combined with 60% RRM; the animals had to be terminated after 5 weeks. In only 60% of animals with 40 mg/kg BW STZ plus 60% RRM was IDDM produced. On the other hand, 65 mg/kg BW STZ in rats with 25% RRM regularly produced IDDM and hypertension without excessive loss of body weight. In these rats, albuminuria developed in 2 weeks. Extracellular fluid volume was elevated and plasma renin activity was depressed. The animals were healthy and hypertensive when killed at the 13th week. We suggest that the 25% RRM rat receiving 65 mg/kg BW STZ is a consistent model of IDDM hypertension, which may be useful in probing the mechanism of this type of hypertension. Am J Hypertens 1992;5:671-680. KEY WORDS: Hypertension, diabetes mellitus, streptozotocin, reduced renal mass, rat.
model have been unsuccessful. " This paper describes the results of our attempts to develop such a model. In IDDM, blood pressure tends to rise when albuminuria becomes apparent and IDDM hypertension is frequently associated with diabetic nephropathy. " Rats receiving streptozotocin (STZ) regularly develop diabetes but irregularly develop hypertension and do not develop nephropathy. We reasoned that STZ might regularly produce hypertension in the presence of impaired renal function. We, therefore, superimposed STZ on two degrees of surgical reduction of renal mass (RRM) that by themselves do not raise blood pressure in rats. We found one combination of STZ and RRM which regularly raises blood pressure while the animal remains healthy. 4
9
10
13
14
Received January 30, 1992. Accepted June 16, 1992. From the Department of Physiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland. Preliminary reports of this data appeared in FASEB J 1989;3:A1316; FASEB J 1990;4:A859; and Physiologist 1990;33:A54. Address correspondence and reprint requests to Motilal B. Pamnani, MBBS, PhD, Department of Physiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799.
© 1992 by the American Journal of Hypertension, Inc.
0895-7061/'92/'$5.00
Downloaded from https://academic.oup.com/ajh/article-abstract/5/10/671/179652 by Columbia University Libraries user on 18 January 2019
A Consistent Model of Insulin-Dependent Diabetes Mellitus Hypertension
672
CHEN ET AL
MATERIALS AND M E T H O D S
Preparation of 60% Reduction in Renal Mass Surgical reduction in renal mass was produced as previously de scribed. Briefly, under ether anesthesia, through a midabdominal incision, the right kidney ( 5 0 % of the total renal mass) and one pole of the left kidney ( 1 0 % of the total renal mass; this calculation is based on the assumption that the two kidneys are approximately equal in weight) were removed. The latter was achieved by encircling the pole with a loop of No. 4-0 silk suture and then tightening the loop. This method both cuts the tissue and ties off the arteries and veins in the excised area. Following recovery from the surgery, all animals consumed normal rat chow and drank tap water ad libitum and were placed individually in metabolic cages. Systolic blood pressure and body weights were recorded weekly. Food and water intake and urine volume were measured daily. Urinary glucose, protein, and albumin excretion were monitored weekly. After 1 week of con trol measurements and documented normotension, rats were randomly divided into experimental (E) and con trol (C) groups. 15,16
Injection ofSTZ in Ε Rats, 60 RRM/65 STZ Under ether anesthesia and sterile conditions, Ε rats received a single bolus injection into the external jugular vein of STZ (Sigma, St. Louis, MO): 65 mg/kg body weight (BW) freshly dissolved in citrate-buffered saline solution, (isosmotic, pH 4.5), in a volume of 1 mL/kg BW. The C group rats, similarly prepared, received an equal volume injection of the vehicle, citrate-buffered saline only (60 RRM/65 V rats). Following injection of STZ or vehicle, animals were allowed to recover from the anesthesia and all the parameters were measured as during the control period for 5 weeks. Preliminary results showed that the 6 0 % RRM rats treated with 65 mg/kg BW STZ developed diabetes mellitus. While they also developed hypertension, the hypertension was not sustained beyond 5 weeks and the rats rapidly lost body weight and muscle mass. We there fore examined two other models of STZ-induced dia betes in reduced renal mass rats. In one model, we kept the reduction of renal mass the same, ie, 6 0 % , but re duced the dose of STZ from 65 mg to 40 mg/kg BW (60
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RRM/40 STZ rats). In the second model, we kept the dose of STZ the same, ie, 65 mg/kg BW, but reduced renal mass by only 2 5 % (25 RRM/65 STZ rats). Also, we injected 65 mg/kg BW STZ in normal rats (2K/65 STZ rats). 60 RRM/40 STZ Rats Animals were individually placed in the metabolic cages and all parameters measured during the 1-week control period as described for the first series of experiments (60 RRM/65 STZ rats). At the end of the control period, the rats were again divided into Ε and C group rats. Under ether anesthesia, Ε rats received a bolus injection of 40 mg/kg BW STZ into the right external jugular vein (60 RRM/40 STZ rats). The C rats were treated in the same manner, except they re ceived a bolus injection of vehicle only (60 RRM/40 V rats). Following injection of STZ or vehicle, blood pres sure and body weight and urinary excretion of glucose, protein, and albumin were monitored weekly and food and water intake and urine volume were monitored daily for 13 weeks. 25 RRM/65 STZ Rats The protocol was again the same as in the first series of experiments (60 RRM/65 STZ rats), except that these animals underwent only 2 5 % reduction in renal mass. This was achieved by removing the lower pole of both kidneys, each weighing about 12 to 1 3 % of the total renal mass (these calculations are based on our data base of approximately 1000 kidney weights in this age and weight group), using loops of silk suture as described above. Following surgery, control measurements were again made for 1 week. At the end of this period, Ε rats received 65 mg/kg BW STZ (25 RRM/65 STZ) and C rats were given vehicle treatment (25 RRM/65 V rats). The monitoring protocol was the same as for the 60 RRM/40 STZ rats. 2K/65 STZ Rats The untouched normal Wistar rats, which had matched age and body weight, received a bolus injection of 65 mg/kg BW STZ as in the first series (60 RRM/65 STZ). Following injection the protocol for monitoring was the same as in 60 RRM/40 STZ rats. Measurement of Blood Pressure At the end of the observing period, animals were anesthetized with pen tobarbital (50 mg/kg BW) and blood pressure and heart rate were recorded directly through a carotid artery catheter using a pressure transducer (Model P23GP, Gould Statham, Oxford, CA) coupled to a Hewlett Packard recorder (Model 7758B, Palo Alto CA). Follow ing the blood pressure measurement, a blood sample (0.2 mL) was removed for measurement of blood pH. Next, 3.0 mL of blood was removed for the measure ment of plasma sodium, potassium, chloride, calcium, magnesium, blood urea nitrogen (BUN), glucose, creati nine, and protein. Hearts and kidneys were then re moved for measurement of heart and kidney weights and heart and kidney to body weight ratios.
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Animals Male Wistar rats (100 to 150 g) were pur chased from Charles River Laboratories, Inc. (Wilming ton, MA) and were placed on a standard rat chow and tap water ad libitum. These animals were housed in a humidity- and temperature-controlled room with 12-h light and dark cycles. One week after receiving the ani mals, systolic blood pressure was recorded by tail pleth ysmography (Natsume KN 209, Tokyo, Japan) weekly to document normotension. When animals reached 250 to 300 g body weight, they underwent reduction in renal mass.
AJH-OCTOBER
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MODEL OF DIABETIC HYPERTENSION
5, NO. 10
17
30
Measurement and Plasma Composition Sodium and potassium concentrations were measured by flame photometry (Beckman, Klina Flame, Palo Alto, C A), cal cium and magnesium concentrations by atomic absorp tion spectrophotometry (Perkin Elmer 603, Norwalk, CT), chloride by chloride titrator (Radiometer A/S, Co penhagen), osmolality by vapor pressure osmometry (Wescor Inc., Model 51000B, Logan, UT), creatinine by spectrophotometric (Beckman spectrophotometer Model #26, Palo Alto, CA) method, blood urea nitro gen (BUN) by colorimetry (Sigma, St. Louis, MO), total protein by the Biuret method, and glucose with a Beck man Glucose Analyzer. 18
Measurement of Urinary Albumin Excretion Twenty-four-hour urinary albumin excretion was de termined using an enzyme-linked immunoabsorbent assay based on the method of Fielding, Price, and Houlton. Polystyrene plates (Corning, Corning, NY) were coated with 1 : 1 0 0 0 goat IgG to rat albumin (Cappell Laboratories, Durham, NC) in phosphate-buffered sa line (PBS) at pH 7.4. The plates were incubated at 4°C for 18 h, washed with 0 . 0 5 % Tween-40 in PBS (PBSTween), and blocked with 0 . 5 % swine skin gelatin in PBS (PBS-SSG). Rat albumin standards (Sigma) were made by serial dilution in PBS-SSG. Rat urine samples from 24-h collections in metabolic cages also were in PBS-SSG. Diluted urine or standards were placed in the wells, and incubated at room temperature for 1 h. After incubation and washing, 1 : 8 0 0 0 rabbit IgG to rat albu min conjugated with peroxidase (Cappell Laboratories) in PBS-SSG was added to each well. Plates were incu bated at room temperature for 1 h and washed and de 19
veloped using 1 : 1 A B C S : H 0 (Kierkgaard-Perry Labo ratories, Gaithersburg, MD). The absorbance was read at 405 nm using a microliter plate reader (Titretek, Flow Laboratories, McLean, VA). 2
2
Data Analysis Student's t test was used to compare means of independent samples. Analysis of variance with repeated measure (TSRM) followed by Duncan's multiple range test was used for among group compari son. A Ρ < .05 was considered significant. RESULTS 60 RRM/65 STZ Rats As expected, following bolus injection of STZ, the Ε rats developed diabetes mellitus as indicated by polyuria, glycosuria (Table 1), and hyper glycemia ( > 5 0 0 mg/dL) (Table 2). Additionally, all 60 RRM/65 STZ rats developed a progressive increase in blood pressure from 119.0 ± 1.4 to 157.0 ± 2.2 mm Hg of systolic pressure during the first 3 weeks, following STZ injection (Figure 1). Thereafter the blood pressure tended to decline. By the 5th week, the body weight had decreased by 1 9 % compared to control body weights before STZ injection and muscle wasting was so exten sive that the animals had to be killed. The Ε rats showed a significant decrease in the plasma levels of sodium and chloride, but increased levels of potassium, magnesium, and creatinine (Table 2). The plasma calcium levels were not significantly different; the plasma osmolality of the Ε rats was higher than that of C rats (Table 2). 60 RRM/40 STZ and 25 RRM/65 STZ Rats Only seven of twelve 6 0 % RRM rats receiving 40 mg/kg BW STZ developed diabetes mellitus (60%) and all seven also developed a progressive increase in systolic blood pressure from 118 ± 1.3 to 160 ± 3.9 mmHg (Figure 1). In contrast, all of the 2 5 % RRM rats receiving 65 mg/kg BW STZ developed diabetes and also developed a pro gressive increase in blood pressure from 119 ± 1.3 to 164 ± 3.2 mm Hg (Figure 1). The increased blood pres sure in both groups was sustained for as long as 13 weeks when the blood pressure was confirmed by direct measurement (Table 3). However, their respective con trols, namely, 60 RRM/40 V and 25 RRM/65 V rats, remained normotensive (Table 3), even though they had a mild terminal increase in systolic blood pressure de termined by plethysmography (Figure 1). Heart rate de creased in all RRM diabetic groups, but was only statis tically significant in the 60 RRM/65 STZ group (Table 3). Unlike the animals in the first series (60 RRM/65 STZ rats), both groups showed a steady increase in body weight over the 13-week period, although at a slower rate than their respective controls (Figure 2). In both 60 RRM/40 STZ and 25 RRM/65 STZ rats, diabetes and hypertension were associated with an increase in left ventricle to body weight ratio and kidney to body weight ratio (Table 3). Plasma sodium did not decrease as it did in 60 RRM/65 STZ rats, whereas calcium de-
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Plasma Renin Activity and Extracellular Fluid Vol ume Some 25 RRM/65 STZ, 25 RRM/65 V, and 2K/65 STZ rats and their respective controls were used exclu sively to measure plasma renin activity and extracellular fluid volumes at 3 weeks after STZ or vehicle treatment. Plasma renin activity was measured by the method of Sealey and Laragh using a commercially available kit (Baxter, MA). Sodium thiocyanate (Sigma) was used to determine extracellular fluid volume. Under pento barbital sodium anesthesia (50 mg/kg BW) 0.5 mL of arterial blood was removed through the left carotid ar tery, spun for 1 min in a microcentrifuge, and 0.25 mL of plasma removed for determination of background absorbance values. The cells were resuspended in 0.25 mL of normal saline and returned to the animal. The urinary bladder was cannulated and a urine sample collected for determination of background absorbance. The urinary bladder was then emptied and 0.5 mL of 1 % sodium thiocyanate solution was injected via the jugular vein cannula. Blood and urine were collected for determination of sodium thiocyanate concentration by spectrophotometry at 490 nm 60 min after the sodium thiocyanate injection.
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CHEN ET AL
1992-VOL
5, NO. 10
TABLE 1. URINE AND URINARY GLUCOSE EXCRETION OF REDUCED RENAL MASS AND NORMAL RATS TREATED WITH STREPTOZOTOCIN (STZ) OR VEHICLE (V) Control
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Week 5
Week 9
Week 13
uv
Uglu
UV
Uglu
UV
Uglu
UV
Uglu
UV
Uglu
34.5 ±2.1 32.0 ±7.5 27.3 ± 1.6 29.0 ± 1.6 26.5 ±2.0 27.6 ±1.1 28.5 ±2.5
0.008 ± 0.001 0.009 ±0.001 0.006 ± 0.001 0.005 ± 0.001 0.007 ±0.001 0.005 ± 0.001 0.007 ± 0.003
160* ±13.5 27.3 ±2.7 157* ±29.4 26.1 ± 1.3 161* ±18 22.8 ±1.8 122* ± 11
9.8* ±0.8 0.007 ± 0.001 9.4* ±0.5 0.005 ± 0.001 10.6* ±1.1 0.005 ± 0.001 9.0* ±0.9
154* ± 13.1 39.1 ±3.8 146* ± 10.9 35.9 ±2.6 167* ± 11 30.1 ±2.6 121* ±13
7.9* ±0.8 0.009 ± 0.001 9.7* ±0.7 0.007 ± 0.001 10.5* ±0.9 0.007 ± 0.001 9.2* ±0.9
161* ±2.5 31.5 ±2.0 180* ±7.2 25.3 ±3.0 155* ±9.7
8.5* ±0.6 0.007 ± 0.001 9.7* ±1.2 0.005 ±0.001 9.7* ±0.5
159* ±4.8 33.5 ±2.1 181* ±4.2 26.2 ±2.1 158* ±9.6
7.5* ±0.6 0.006 ± 0.001 9.5* ±0.7 0.006 ± 0.001 9.8* ±0.7
* Ρ < .05 differences from respective control periods. UV: urine volume (mL/24 h); Uglu: urinary glucose excretion (g/24 h).
creased relative to controls. The other changes in the plasma composition were the same as described for the 60 RRM/65 STZ rats, ie, the plasma chloride decreased and potassium, magnesium, and creatinine increased (Table 2). With analysis of acid-base profile (data not shown), decreased blood pH in both diabetic groups showed metabolic acidosis (Table 2). Urinary total pro tein (Figure 3) and albumin excretion (Figure 4) in creased in 60 RRM/40 STZ rats by the 2nd and 1st week, respectively, following STZ injection. The increase in albumin excretion preceded the increase in blood pres sure. In 25 RRM/65 STZ rats, the total protein and albu min excretion were significantly elevated by the 4th (Fig ure 3) and 2nd week (Figure 4) post STZ injection, respectively. The increase in albumin excretion was temporally correlated to the increase in blood pressure (Figures 1, 4). The 60 RRM/40 V rats had increased urinary protein and albumin excretion at the 6th and 5th week, respectively, following reduction in renal mass. However, in the 25 RRM/65 V animals protein and albumin excretion remained normal throughout the study (Figures 3, 4). Volume studies performed at 3 weeks following STZ treatment showed significant increased intracellular fluid volume in hypertensive 25 RRM/65 STZ com pared with normotensive 25 RRM/65 V and 2K/65 STZ rats (Table 4). In addition, 25 RRM/65 STZ rats had a more depressed plasma renin activity level than 2K/65 STZ rats (Table 4). 2K/65 STZ Rats All normal rats developed diabetes following a 65 mg/kg BW STZ injection. Blood pressure increased slightly by plethysmography at 5 weeks post treatment (Figure 1), but was normal by direct measure
ment at termination (Table 3). Total protein excretion remained normal until 12 weeks after STZ treatment, while urinary albumin excretion was increased at the 6th week following STZ-induced IDDM. DISCUSSION The data show that STZ-induced IDDM rats with both 2 5 % and 6 0 % reduction in renal mass develop hyper tension. The plasma composition in diabetic 60 RRM/ 40 STZ and 25 RRM/65 STZ is consistent with uncon trolled diabetes mellitus, namely, hyperglycemia, hyperosmolality, and metabolic acidosis. Hyperten sion does not develop when these animals fail to be come diabetic ( 4 0 % of the 60 RRM/40 STZ rats) or on injection of the vehicle in control rats with the same degree of reduction in renal mass, demonstrating that 2 5 % to 6 0 % reduction in renal mass, by itself, does not induce hypertension. This confirms our previous find ings that 6 0 % RRM in rats consuming a salt-free diet and 1 % saline or consuming a normal rat diet and tap water (unpublished observation) remain normotensive. Any further reduction in renal mass while drinking sa line induces hypertension in these rats. These obser vations suggest that an additional factor is necessary, in this case IDDM, to induce hypertension in rats with reduction in renal mass of 6 0 % or less. On the other hand, the RRM seems to be necessary for the acute or subacute development of hypertension in IDDM rats. Hypertension does not fully develop when IDDM is induced by STZ in normal Wistar rats (2K/65 STZ). Blood pressure did not increase for 5 weeks after STZ treatment whereas it increased almost immediately in the 25 RRM/65 STZ group. Furthermore, after 5 weeks, blood pressure did not increase to the same ex20
15
15
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60RRM/65STZ (N = 10) 60RRM/65V (N = 9) 60RRM/40STZ (N = 6) 60RRM/40V (N = 6) 25RRM/65STZ (N = 10) 25RRM/65V (N = 7) 2K/65STZ (N = 7)
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AJH-OCTOBER
1992-VOL
5, NO. 10
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1992;5:671-680
ORIGINAL CONTRIBUTIONS
S. Chen, C. Yuan, J.F. Schooley, Jr., F.J. Haddy, and M.B. Pamnani
Hypertension is frequently seen in insulindependent diabetes mellitus (IDDM), but the mechanism of the hypertension is unknown. An animal model of IDDM hypertension could be helpful in determining the mechanism, but experimental IDDM has been infrequently and irregularly associated with hypertension. In an attempt to develop a consistent model of IDDM hypertension, we superimposed streptozotocin (STZ)-induced IDDM on surgical reduction of renal mass (RRM) in Wistar rats. Seven groups of rats were studied: 1) 60% RRM receiving 65 mg/kg body weight (BW) STZ; 2) 60% RRM receiving 40 mg/kg BW STZ; 3) 25% RRM receiving 65 mg/kg BW STZ; 4) two kidney normal rats receiving 65 mg/kg BW STZ; 5) 60% RRM receiving vehicle (control for group 1); 6) 60% RRM receiving vehicle (control for group 2); and 7) 25% RRM receiving vehicle. STZ produced diabetes and hypertension within 1 to 2 weeks in all three group's of RRM rats but blood pressure was unaffected by 60% or 25% RRM alone. STZ alone had no
H
ypertension is frequently seen in insulindependent diabetes mellitus (IDDM). " The mechanism of this hypertension is unknown. An animal model of IDDM hypertension might be useful in probing the mechanism of this type of hypertension but attempts to develop such a consistent 1
3
effect on blood pressure until the 5th week when the blood pressure increased slightly. Progressive weight loss resulted from 65 mg/kg BW STZ combined with 60% RRM; the animals had to be terminated after 5 weeks. In only 60% of animals with 40 mg/kg BW STZ plus 60% RRM was IDDM produced. On the other hand, 65 mg/kg BW STZ in rats with 25% RRM regularly produced IDDM and hypertension without excessive loss of body weight. In these rats, albuminuria developed in 2 weeks. Extracellular fluid volume was elevated and plasma renin activity was depressed. The animals were healthy and hypertensive when killed at the 13th week. We suggest that the 25% RRM rat receiving 65 mg/kg BW STZ is a consistent model of IDDM hypertension, which may be useful in probing the mechanism of this type of hypertension. Am J Hypertens 1992;5:671-680. KEY WORDS: Hypertension, diabetes mellitus, streptozotocin, reduced renal mass, rat.
model have been unsuccessful. " This paper describes the results of our attempts to develop such a model. In IDDM, blood pressure tends to rise when albuminuria becomes apparent and IDDM hypertension is frequently associated with diabetic nephropathy. " Rats receiving streptozotocin (STZ) regularly develop diabetes but irregularly develop hypertension and do not develop nephropathy. We reasoned that STZ might regularly produce hypertension in the presence of impaired renal function. We, therefore, superimposed STZ on two degrees of surgical reduction of renal mass (RRM) that by themselves do not raise blood pressure in rats. We found one combination of STZ and RRM which regularly raises blood pressure while the animal remains healthy. 4
9
10
13
14
Received January 30, 1992. Accepted June 16, 1992. From the Department of Physiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland. Preliminary reports of this data appeared in FASEB J 1989;3:A1316; FASEB J 1990;4:A859; and Physiologist 1990;33:A54. Address correspondence and reprint requests to Motilal B. Pamnani, MBBS, PhD, Department of Physiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799.
© 1992 by the American Journal of Hypertension, Inc.
0895-7061/'92/'$5.00
Downloaded from https://academic.oup.com/ajh/article-abstract/5/10/671/179652 by Columbia University Libraries user on 18 January 2019
A Consistent Model of Insulin-Dependent Diabetes Mellitus Hypertension
672
CHEN ET AL
MATERIALS AND M E T H O D S
Preparation of 60% Reduction in Renal Mass Surgical reduction in renal mass was produced as previously de scribed. Briefly, under ether anesthesia, through a midabdominal incision, the right kidney ( 5 0 % of the total renal mass) and one pole of the left kidney ( 1 0 % of the total renal mass; this calculation is based on the assumption that the two kidneys are approximately equal in weight) were removed. The latter was achieved by encircling the pole with a loop of No. 4-0 silk suture and then tightening the loop. This method both cuts the tissue and ties off the arteries and veins in the excised area. Following recovery from the surgery, all animals consumed normal rat chow and drank tap water ad libitum and were placed individually in metabolic cages. Systolic blood pressure and body weights were recorded weekly. Food and water intake and urine volume were measured daily. Urinary glucose, protein, and albumin excretion were monitored weekly. After 1 week of con trol measurements and documented normotension, rats were randomly divided into experimental (E) and con trol (C) groups. 15,16
Injection ofSTZ in Ε Rats, 60 RRM/65 STZ Under ether anesthesia and sterile conditions, Ε rats received a single bolus injection into the external jugular vein of STZ (Sigma, St. Louis, MO): 65 mg/kg body weight (BW) freshly dissolved in citrate-buffered saline solution, (isosmotic, pH 4.5), in a volume of 1 mL/kg BW. The C group rats, similarly prepared, received an equal volume injection of the vehicle, citrate-buffered saline only (60 RRM/65 V rats). Following injection of STZ or vehicle, animals were allowed to recover from the anesthesia and all the parameters were measured as during the control period for 5 weeks. Preliminary results showed that the 6 0 % RRM rats treated with 65 mg/kg BW STZ developed diabetes mellitus. While they also developed hypertension, the hypertension was not sustained beyond 5 weeks and the rats rapidly lost body weight and muscle mass. We there fore examined two other models of STZ-induced dia betes in reduced renal mass rats. In one model, we kept the reduction of renal mass the same, ie, 6 0 % , but re duced the dose of STZ from 65 mg to 40 mg/kg BW (60
1992-VOL
5, NO. 10
RRM/40 STZ rats). In the second model, we kept the dose of STZ the same, ie, 65 mg/kg BW, but reduced renal mass by only 2 5 % (25 RRM/65 STZ rats). Also, we injected 65 mg/kg BW STZ in normal rats (2K/65 STZ rats). 60 RRM/40 STZ Rats Animals were individually placed in the metabolic cages and all parameters measured during the 1-week control period as described for the first series of experiments (60 RRM/65 STZ rats). At the end of the control period, the rats were again divided into Ε and C group rats. Under ether anesthesia, Ε rats received a bolus injection of 40 mg/kg BW STZ into the right external jugular vein (60 RRM/40 STZ rats). The C rats were treated in the same manner, except they re ceived a bolus injection of vehicle only (60 RRM/40 V rats). Following injection of STZ or vehicle, blood pres sure and body weight and urinary excretion of glucose, protein, and albumin were monitored weekly and food and water intake and urine volume were monitored daily for 13 weeks. 25 RRM/65 STZ Rats The protocol was again the same as in the first series of experiments (60 RRM/65 STZ rats), except that these animals underwent only 2 5 % reduction in renal mass. This was achieved by removing the lower pole of both kidneys, each weighing about 12 to 1 3 % of the total renal mass (these calculations are based on our data base of approximately 1000 kidney weights in this age and weight group), using loops of silk suture as described above. Following surgery, control measurements were again made for 1 week. At the end of this period, Ε rats received 65 mg/kg BW STZ (25 RRM/65 STZ) and C rats were given vehicle treatment (25 RRM/65 V rats). The monitoring protocol was the same as for the 60 RRM/40 STZ rats. 2K/65 STZ Rats The untouched normal Wistar rats, which had matched age and body weight, received a bolus injection of 65 mg/kg BW STZ as in the first series (60 RRM/65 STZ). Following injection the protocol for monitoring was the same as in 60 RRM/40 STZ rats. Measurement of Blood Pressure At the end of the observing period, animals were anesthetized with pen tobarbital (50 mg/kg BW) and blood pressure and heart rate were recorded directly through a carotid artery catheter using a pressure transducer (Model P23GP, Gould Statham, Oxford, CA) coupled to a Hewlett Packard recorder (Model 7758B, Palo Alto CA). Follow ing the blood pressure measurement, a blood sample (0.2 mL) was removed for measurement of blood pH. Next, 3.0 mL of blood was removed for the measure ment of plasma sodium, potassium, chloride, calcium, magnesium, blood urea nitrogen (BUN), glucose, creati nine, and protein. Hearts and kidneys were then re moved for measurement of heart and kidney weights and heart and kidney to body weight ratios.
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Animals Male Wistar rats (100 to 150 g) were pur chased from Charles River Laboratories, Inc. (Wilming ton, MA) and were placed on a standard rat chow and tap water ad libitum. These animals were housed in a humidity- and temperature-controlled room with 12-h light and dark cycles. One week after receiving the ani mals, systolic blood pressure was recorded by tail pleth ysmography (Natsume KN 209, Tokyo, Japan) weekly to document normotension. When animals reached 250 to 300 g body weight, they underwent reduction in renal mass.
AJH-OCTOBER
AJH-OCTOBER
1992-VOL
MODEL OF DIABETIC HYPERTENSION
5, NO. 10
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Measurement and Plasma Composition Sodium and potassium concentrations were measured by flame photometry (Beckman, Klina Flame, Palo Alto, C A), cal cium and magnesium concentrations by atomic absorp tion spectrophotometry (Perkin Elmer 603, Norwalk, CT), chloride by chloride titrator (Radiometer A/S, Co penhagen), osmolality by vapor pressure osmometry (Wescor Inc., Model 51000B, Logan, UT), creatinine by spectrophotometric (Beckman spectrophotometer Model #26, Palo Alto, CA) method, blood urea nitro gen (BUN) by colorimetry (Sigma, St. Louis, MO), total protein by the Biuret method, and glucose with a Beck man Glucose Analyzer. 18
Measurement of Urinary Albumin Excretion Twenty-four-hour urinary albumin excretion was de termined using an enzyme-linked immunoabsorbent assay based on the method of Fielding, Price, and Houlton. Polystyrene plates (Corning, Corning, NY) were coated with 1 : 1 0 0 0 goat IgG to rat albumin (Cappell Laboratories, Durham, NC) in phosphate-buffered sa line (PBS) at pH 7.4. The plates were incubated at 4°C for 18 h, washed with 0 . 0 5 % Tween-40 in PBS (PBSTween), and blocked with 0 . 5 % swine skin gelatin in PBS (PBS-SSG). Rat albumin standards (Sigma) were made by serial dilution in PBS-SSG. Rat urine samples from 24-h collections in metabolic cages also were in PBS-SSG. Diluted urine or standards were placed in the wells, and incubated at room temperature for 1 h. After incubation and washing, 1 : 8 0 0 0 rabbit IgG to rat albu min conjugated with peroxidase (Cappell Laboratories) in PBS-SSG was added to each well. Plates were incu bated at room temperature for 1 h and washed and de 19
veloped using 1 : 1 A B C S : H 0 (Kierkgaard-Perry Labo ratories, Gaithersburg, MD). The absorbance was read at 405 nm using a microliter plate reader (Titretek, Flow Laboratories, McLean, VA). 2
2
Data Analysis Student's t test was used to compare means of independent samples. Analysis of variance with repeated measure (TSRM) followed by Duncan's multiple range test was used for among group compari son. A Ρ < .05 was considered significant. RESULTS 60 RRM/65 STZ Rats As expected, following bolus injection of STZ, the Ε rats developed diabetes mellitus as indicated by polyuria, glycosuria (Table 1), and hyper glycemia ( > 5 0 0 mg/dL) (Table 2). Additionally, all 60 RRM/65 STZ rats developed a progressive increase in blood pressure from 119.0 ± 1.4 to 157.0 ± 2.2 mm Hg of systolic pressure during the first 3 weeks, following STZ injection (Figure 1). Thereafter the blood pressure tended to decline. By the 5th week, the body weight had decreased by 1 9 % compared to control body weights before STZ injection and muscle wasting was so exten sive that the animals had to be killed. The Ε rats showed a significant decrease in the plasma levels of sodium and chloride, but increased levels of potassium, magnesium, and creatinine (Table 2). The plasma calcium levels were not significantly different; the plasma osmolality of the Ε rats was higher than that of C rats (Table 2). 60 RRM/40 STZ and 25 RRM/65 STZ Rats Only seven of twelve 6 0 % RRM rats receiving 40 mg/kg BW STZ developed diabetes mellitus (60%) and all seven also developed a progressive increase in systolic blood pressure from 118 ± 1.3 to 160 ± 3.9 mmHg (Figure 1). In contrast, all of the 2 5 % RRM rats receiving 65 mg/kg BW STZ developed diabetes and also developed a pro gressive increase in blood pressure from 119 ± 1.3 to 164 ± 3.2 mm Hg (Figure 1). The increased blood pres sure in both groups was sustained for as long as 13 weeks when the blood pressure was confirmed by direct measurement (Table 3). However, their respective con trols, namely, 60 RRM/40 V and 25 RRM/65 V rats, remained normotensive (Table 3), even though they had a mild terminal increase in systolic blood pressure de termined by plethysmography (Figure 1). Heart rate de creased in all RRM diabetic groups, but was only statis tically significant in the 60 RRM/65 STZ group (Table 3). Unlike the animals in the first series (60 RRM/65 STZ rats), both groups showed a steady increase in body weight over the 13-week period, although at a slower rate than their respective controls (Figure 2). In both 60 RRM/40 STZ and 25 RRM/65 STZ rats, diabetes and hypertension were associated with an increase in left ventricle to body weight ratio and kidney to body weight ratio (Table 3). Plasma sodium did not decrease as it did in 60 RRM/65 STZ rats, whereas calcium de-
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Plasma Renin Activity and Extracellular Fluid Vol ume Some 25 RRM/65 STZ, 25 RRM/65 V, and 2K/65 STZ rats and their respective controls were used exclu sively to measure plasma renin activity and extracellular fluid volumes at 3 weeks after STZ or vehicle treatment. Plasma renin activity was measured by the method of Sealey and Laragh using a commercially available kit (Baxter, MA). Sodium thiocyanate (Sigma) was used to determine extracellular fluid volume. Under pento barbital sodium anesthesia (50 mg/kg BW) 0.5 mL of arterial blood was removed through the left carotid ar tery, spun for 1 min in a microcentrifuge, and 0.25 mL of plasma removed for determination of background absorbance values. The cells were resuspended in 0.25 mL of normal saline and returned to the animal. The urinary bladder was cannulated and a urine sample collected for determination of background absorbance. The urinary bladder was then emptied and 0.5 mL of 1 % sodium thiocyanate solution was injected via the jugular vein cannula. Blood and urine were collected for determination of sodium thiocyanate concentration by spectrophotometry at 490 nm 60 min after the sodium thiocyanate injection.
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AJH-OCTOBER
CHEN ET AL
1992-VOL
5, NO. 10
TABLE 1. URINE AND URINARY GLUCOSE EXCRETION OF REDUCED RENAL MASS AND NORMAL RATS TREATED WITH STREPTOZOTOCIN (STZ) OR VEHICLE (V) Control
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Week 5
Week 9
Week 13
uv
Uglu
UV
Uglu
UV
Uglu
UV
Uglu
UV
Uglu
34.5 ±2.1 32.0 ±7.5 27.3 ± 1.6 29.0 ± 1.6 26.5 ±2.0 27.6 ±1.1 28.5 ±2.5
0.008 ± 0.001 0.009 ±0.001 0.006 ± 0.001 0.005 ± 0.001 0.007 ±0.001 0.005 ± 0.001 0.007 ± 0.003
160* ±13.5 27.3 ±2.7 157* ±29.4 26.1 ± 1.3 161* ±18 22.8 ±1.8 122* ± 11
9.8* ±0.8 0.007 ± 0.001 9.4* ±0.5 0.005 ± 0.001 10.6* ±1.1 0.005 ± 0.001 9.0* ±0.9
154* ± 13.1 39.1 ±3.8 146* ± 10.9 35.9 ±2.6 167* ± 11 30.1 ±2.6 121* ±13
7.9* ±0.8 0.009 ± 0.001 9.7* ±0.7 0.007 ± 0.001 10.5* ±0.9 0.007 ± 0.001 9.2* ±0.9
161* ±2.5 31.5 ±2.0 180* ±7.2 25.3 ±3.0 155* ±9.7
8.5* ±0.6 0.007 ± 0.001 9.7* ±1.2 0.005 ±0.001 9.7* ±0.5
159* ±4.8 33.5 ±2.1 181* ±4.2 26.2 ±2.1 158* ±9.6
7.5* ±0.6 0.006 ± 0.001 9.5* ±0.7 0.006 ± 0.001 9.8* ±0.7
* Ρ < .05 differences from respective control periods. UV: urine volume (mL/24 h); Uglu: urinary glucose excretion (g/24 h).
creased relative to controls. The other changes in the plasma composition were the same as described for the 60 RRM/65 STZ rats, ie, the plasma chloride decreased and potassium, magnesium, and creatinine increased (Table 2). With analysis of acid-base profile (data not shown), decreased blood pH in both diabetic groups showed metabolic acidosis (Table 2). Urinary total pro tein (Figure 3) and albumin excretion (Figure 4) in creased in 60 RRM/40 STZ rats by the 2nd and 1st week, respectively, following STZ injection. The increase in albumin excretion preceded the increase in blood pres sure. In 25 RRM/65 STZ rats, the total protein and albu min excretion were significantly elevated by the 4th (Fig ure 3) and 2nd week (Figure 4) post STZ injection, respectively. The increase in albumin excretion was temporally correlated to the increase in blood pressure (Figures 1, 4). The 60 RRM/40 V rats had increased urinary protein and albumin excretion at the 6th and 5th week, respectively, following reduction in renal mass. However, in the 25 RRM/65 V animals protein and albumin excretion remained normal throughout the study (Figures 3, 4). Volume studies performed at 3 weeks following STZ treatment showed significant increased intracellular fluid volume in hypertensive 25 RRM/65 STZ com pared with normotensive 25 RRM/65 V and 2K/65 STZ rats (Table 4). In addition, 25 RRM/65 STZ rats had a more depressed plasma renin activity level than 2K/65 STZ rats (Table 4). 2K/65 STZ Rats All normal rats developed diabetes following a 65 mg/kg BW STZ injection. Blood pressure increased slightly by plethysmography at 5 weeks post treatment (Figure 1), but was normal by direct measure
ment at termination (Table 3). Total protein excretion remained normal until 12 weeks after STZ treatment, while urinary albumin excretion was increased at the 6th week following STZ-induced IDDM. DISCUSSION The data show that STZ-induced IDDM rats with both 2 5 % and 6 0 % reduction in renal mass develop hyper tension. The plasma composition in diabetic 60 RRM/ 40 STZ and 25 RRM/65 STZ is consistent with uncon trolled diabetes mellitus, namely, hyperglycemia, hyperosmolality, and metabolic acidosis. Hyperten sion does not develop when these animals fail to be come diabetic ( 4 0 % of the 60 RRM/40 STZ rats) or on injection of the vehicle in control rats with the same degree of reduction in renal mass, demonstrating that 2 5 % to 6 0 % reduction in renal mass, by itself, does not induce hypertension. This confirms our previous find ings that 6 0 % RRM in rats consuming a salt-free diet and 1 % saline or consuming a normal rat diet and tap water (unpublished observation) remain normotensive. Any further reduction in renal mass while drinking sa line induces hypertension in these rats. These obser vations suggest that an additional factor is necessary, in this case IDDM, to induce hypertension in rats with reduction in renal mass of 6 0 % or less. On the other hand, the RRM seems to be necessary for the acute or subacute development of hypertension in IDDM rats. Hypertension does not fully develop when IDDM is induced by STZ in normal Wistar rats (2K/65 STZ). Blood pressure did not increase for 5 weeks after STZ treatment whereas it increased almost immediately in the 25 RRM/65 STZ group. Furthermore, after 5 weeks, blood pressure did not increase to the same ex20
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60RRM/65STZ (N = 10) 60RRM/65V (N = 9) 60RRM/40STZ (N = 6) 60RRM/40V (N = 6) 25RRM/65STZ (N = 10) 25RRM/65V (N = 7) 2K/65STZ (N = 7)
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AJH-OCTOBER
1992-VOL
5, NO. 10
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