(1976)
TOXICXKQGYANDAPPLIEDPHARMACOLOGY36,607-610
SHORT The
Effects of Reserpine Catecholamines
COMMUNICATION on Renal Necrosis of Choline-Deficient
and Urinary Rats1
The Effects of Reserpine on Renal Necrosis and Urinary Catecholamines of Choline-Deficient Rats. BRUCE, J. R., WEISE, H. J., AND CARTER, M. K. (1976). Toxicol. Appl. Pharmacol.36, 607-610. Renal necrosis due to a choline-deficient diet was induced in a group of male weanling SpragueDawley rats. A control group wasgiven Purina rat chow, and another group was given a choline chloride supplement (150 mg/lOO ml in tap water) in addition to the choline-de&ient diet. Reserpine, an agent that reduces catecholamines, was given subcutaneously (0.1 mg/kg) daily to one-half of each group. After 16 days the choline-deficient rats given reserpine had significantly less renal necrosis and excreted less catecholamines than choline-deficient rats. Reserpine had no effect on hepatic fatty infiltration due to choline deficiency. The effect of choline deficiency to produce renal hemorrhagic degeneration was reported by Griffith and Wade (1939). The specific mechanism of this degeneration is still being investigated. The present working hypothesis, originally suggested by Wolbach and Bessey (1942) and extended by Baxter (1953), has been suggested to be a neurovascular mechanism. Due to the choline deficiency, there is thought to be an imbaIance between a vasodilator such as acetylcholine and the vasoconstrictor catecholamines. This imbalance caused by a decrease in acetylcholine is postulated to result in vasospasm and thus ischemia. It has been reported by Nagler ef al. (1968) that acetylcholine concentrations in the kidneys of choline-deficient rats were decreased 50-75 % compared to controls. Nagler et al. (1969) also reported that during choline deficiency the vessels of the mesoappendix were much more sensitive to epinephrine than in those animals receiving choline supplements. Therefore, a series of experiments was planned in which the catecholamines would be reduced, and this was accomplished by using reserpine in choline-deficient, cholinesupplemented, and control animals. METHODS Male weanling Sprague-Dawley rats weighing from 43.5 to 51.5 g were obtained from Charles River Lakeview (Netield, New Jersey). Two experiments were carried out. In each experiment the rats were divided into six groups of four and treated as shown in Table 1. The choline-deficient diet was a modification of that given by Wilson et at. (1972) and was prepared in our laboratory. 1 This researchwassupported in part by an NIH Grant No. AM 16080.J. R. Bruce was supported by a Summer Medical Student ResearchFellowship from the Louisiana Heart Association. Inc. Copyright Q 1976 by Academic Press, Inc. All rigbta of reproduction in any form reserved. Printed in Great Britain
607
608
SHORTCOMMUNICATION TABLE
1
EFFECT OF RESERPINE AND CHOLINE-DEFICIENT NECROSIS AND URINARY CATECHOLAMINES
Treatment Control Control
+ R’
CD’ CD+RS CDS’ CDS + Rh
Relative renal necrosis” 0.13 0.21 2.13 1.06 0.38 0.56
-t 0.12 f 0.15 f 0.35 L- 0.41d + 0.16 + 0.18
Total urinary catecholaminesb 0.186 0.151 0.205 0.142 0.281 0.233
+ 0.037 _+ 0.0214 + 0.018 + 0.018d L- 0.043 +- 0.047d
DIET ON RENAL OF RATS
Urinary norepinephrine ti4h-e treat.) 0.160 0.130 0.176 0.122 0.211 0.198
+ 0.033 _+ 0.018’ + 0.015 f 0.015” + 0.015 + 0.041
’ Total scores (0 to +4) per group divided by numbers of rats per group; n = eight rats per group; mean ? SE. b Epinephrine plus norepinephrine; n = 10 urine samples per group. c R = Reserpine, 0.1 mg/kg, SCdaily, 16 days. dp < 0.05 (R groups compared to preceding group). e CD = Choline-deficient diet, 14 days. f CD + R = Choline-deficient diet plus reserpine. g CDS = Choline-deficient diet plus choline chloride supplement (150 mg/lO ml in tap water). * CDS + R = Choline-deficient diet plus choline chloride supplement plus reserpine.
The rats were weighed daily. The appropriate groups received subcutaneous injections of reserpine (Serpasil) beginning 2 days before the choline-deficient diet was begun and continued until the end of the experiment. Excluding the two preliminary days when all rats received Purina chow, the rats were allowed to eat and drink their respective diets ad Iibitum for 14 days. All rats were sacrificed on Day 16. Kidney and liver tissue were taken and sections were stained with hematoxylin and eosin (H & E). The kidney sections (H & E stain) were coded, and a different person graded the slides to prevent the evaluation from being prejudiced. They were graded on a scale from 0 to +4. The grading system used was modeled after that of Levensen et al. (1968). The following criteria were used for grading: 0 = no gross or microscopic pathology; +I = some hyperemia, pycnotic nuclei, and slight focal necrosis of cortex (sloughing of tubular epithelial cells); +2 = more extensive focal necrosis than +l with a few calcium deposits, a few casts, and slight subcapsular hemorrhagic areas; +3 = more extensive than +2 necrosis with moderate number of casts and larger hemorrhagic areas and more calcium deposits; +4 = extensive confluent neurotic areas, many casts extending into medullary areas, increased hemorrhage and number of calcium deposits in necrotic areas. There was no necrosis of glomeruli which would have extended grading to +5 according to Monserrat et al. (1969). Liver sections were also graded using the degree of fatty infiltration. During the period of the 16 days, five 24hr pooled urine samples of each group were obtained on Days 3, 8, 10, 15, and 16. Three milliliters of 1 N HCl were placed in the flasks that were to receive the urine samples to preserve the catecholamines prior to
SHORT
COMMUNICATION
609
assay. The urine samples were analyzed for total catecholamines (norepinephrine and epinephrine) and endogenous urinary creatinine. The catecholamines were analyzed according to the fluorescent hydroxyindole method of Udenfriend (1962). The catecholamine values were factored over the creatinine values to minimize error due to changes in filtration rate. The results of the two experiments were similar so the data were pooled. Student’s t test was used to analyze data for significance at the 5 % level (Snedecor and Cochran, 1967). RESULTS
AND
DISCUSSION
The theory that choline deficiency-induced renal necrosis is caused by an imbalance between vasoconstrictor and vasodilator substances is further substantiated by our results. Reserpine, an agent that reduces catecholamines, caused a decrease in the amount of renal necrosis (Table 1). The amount of necrosis in the CD + R rats was significantly less than that of CD rats. There was a minimal amount of necrosis in the control animals, and there was no significant difference in the degree of necrosis between control and control + R rats. There was also minimal necrosis in CDS and CDS + R. There was no difference in liver sections stained with H & E between CD and CD + R groups. Since reserpine reduced the urinary catecholamines (Table i), this would lessen the difference between the vasoconstrictor catecholamines and the decreased vasodilator acetylcholine, thus perhaps alleviating to some extent vasospasm .and ischemia. Of the total urinary catecholamines, norepinephrine was the major component (Table 1). The CD rats tended to have a higher excretion of urinary catecholamine excretion than did the control animals. In other experiments (unpublished observations) there was a significant increase in urinary catecholamines in choline-deficient rats. In the groups of rats injected with reserpine there was a significantly lower catecholamine excretion than in corresponding groups of rats without reserpine except for the CDS + R group. Although the catecholamine excretion tended to be lower in this group there was not a significant decrease compared to the CDS group with reference to norepinephrine. The correlation of decreased renal necrosis with a decrease in urinary vasoconstrictor catecholamines occasioned by reserpine lends support to the theory that the renal necrosis of choline-deficiency in rats may be due to an imbalance between vasoconstrictor catecholamines and a vasodilator such as acetylcholine. REFERENCES BAXTER, J. (1953). Protective effect of thiouracil and dibenamine against renal injury due to
choline deficiency; Influence of endocrine and autonomic systems. J. Pharmacol. Exp. 7krap. 107,394-401. GRIFFITH, W. H. AND WADE, N. J. (1939). Some effects of low choline diets. Proc. Sot. Exp. Biol. Med. 41, 188-190. LEVENSON, STANLEY M., NAGLER, ARNOLD L., GREEVER, ERVING F. AND SEIFTER, E. (1968).
Acute choline deficiency in germ free, conventionalized and open-animal-room rats: Effects of neomycin, chlortetracycline, vitamin B1z and coproghagy prevention. J. Nutr, 95,247-210.
610
SHORT
COMMUNICATION
A. J., GHOSHAL, A. K., HARTROFT, W. S. AND PORTA, E. A. (1969). Lipoperoxidation in the pathogenesis of renal necrosis in choline-deficient rats. Amer. J. Pathol. 55, 163-189. NAGLER, A. L., BAEZ, S. AND LEVENSON, S. M. (1969). Status of the microcirculation during acute choline deficiency. J. Nutr. 97,232-236. NAGLER, A. L., DETTBARN, W. D., SEIFTER, E. AND LEVENSON, S. M. (1968). Tissue levels of acetylcholine and acetylcholine esterase in weanlingrats subjected to acute choline deficiency. J. Nutr. 94, 13-19. SNEDECOR, G. W. AND C~CHRAN, W. G. (1967). Statistical Methods, 6th ed. The Iowa State University Press, Ames. UDENFRIEND, S. (1962). Tyrosine and its metabolites. In Fluorescence Assay in Biology and Medicine (N. 0. Kaplan and H. A. Scheraga, Eds.), pp. 129-160. Academic Press, New York. WILSON, R. B., HOPPE, H. AND NEWBERNE, P. M. (1972). Hemorrhagic renal necrosis in young adult rats fed a cholesterol supplemented choline deficient diet. Nut. Rep. Infer. 6,275-280. WOLBACH, S. B. AND BESSEY,0. A. (1942). Tissue change in vitamin deficiencies. Physiol. Rev. 22,233-289. J. R. BRUCE H. J. WEISE M. K. CARTER Department of Pharmacology Tulane University School of Medicine New Orleans, Louisiana Received October 7,1975; accepted February IO,1976 MONSERRAT,
TOXICXKQGYANDAPPLIEDPHARMACOLOGY36,607-610
SHORT The
Effects of Reserpine Catecholamines
COMMUNICATION on Renal Necrosis of Choline-Deficient
and Urinary Rats1
The Effects of Reserpine on Renal Necrosis and Urinary Catecholamines of Choline-Deficient Rats. BRUCE, J. R., WEISE, H. J., AND CARTER, M. K. (1976). Toxicol. Appl. Pharmacol.36, 607-610. Renal necrosis due to a choline-deficient diet was induced in a group of male weanling SpragueDawley rats. A control group wasgiven Purina rat chow, and another group was given a choline chloride supplement (150 mg/lOO ml in tap water) in addition to the choline-de&ient diet. Reserpine, an agent that reduces catecholamines, was given subcutaneously (0.1 mg/kg) daily to one-half of each group. After 16 days the choline-deficient rats given reserpine had significantly less renal necrosis and excreted less catecholamines than choline-deficient rats. Reserpine had no effect on hepatic fatty infiltration due to choline deficiency. The effect of choline deficiency to produce renal hemorrhagic degeneration was reported by Griffith and Wade (1939). The specific mechanism of this degeneration is still being investigated. The present working hypothesis, originally suggested by Wolbach and Bessey (1942) and extended by Baxter (1953), has been suggested to be a neurovascular mechanism. Due to the choline deficiency, there is thought to be an imbaIance between a vasodilator such as acetylcholine and the vasoconstrictor catecholamines. This imbalance caused by a decrease in acetylcholine is postulated to result in vasospasm and thus ischemia. It has been reported by Nagler ef al. (1968) that acetylcholine concentrations in the kidneys of choline-deficient rats were decreased 50-75 % compared to controls. Nagler et al. (1969) also reported that during choline deficiency the vessels of the mesoappendix were much more sensitive to epinephrine than in those animals receiving choline supplements. Therefore, a series of experiments was planned in which the catecholamines would be reduced, and this was accomplished by using reserpine in choline-deficient, cholinesupplemented, and control animals. METHODS Male weanling Sprague-Dawley rats weighing from 43.5 to 51.5 g were obtained from Charles River Lakeview (Netield, New Jersey). Two experiments were carried out. In each experiment the rats were divided into six groups of four and treated as shown in Table 1. The choline-deficient diet was a modification of that given by Wilson et at. (1972) and was prepared in our laboratory. 1 This researchwassupported in part by an NIH Grant No. AM 16080.J. R. Bruce was supported by a Summer Medical Student ResearchFellowship from the Louisiana Heart Association. Inc. Copyright Q 1976 by Academic Press, Inc. All rigbta of reproduction in any form reserved. Printed in Great Britain
607
608
SHORTCOMMUNICATION TABLE
1
EFFECT OF RESERPINE AND CHOLINE-DEFICIENT NECROSIS AND URINARY CATECHOLAMINES
Treatment Control Control
+ R’
CD’ CD+RS CDS’ CDS + Rh
Relative renal necrosis” 0.13 0.21 2.13 1.06 0.38 0.56
-t 0.12 f 0.15 f 0.35 L- 0.41d + 0.16 + 0.18
Total urinary catecholaminesb 0.186 0.151 0.205 0.142 0.281 0.233
+ 0.037 _+ 0.0214 + 0.018 + 0.018d L- 0.043 +- 0.047d
DIET ON RENAL OF RATS
Urinary norepinephrine ti4h-e treat.) 0.160 0.130 0.176 0.122 0.211 0.198
+ 0.033 _+ 0.018’ + 0.015 f 0.015” + 0.015 + 0.041
’ Total scores (0 to +4) per group divided by numbers of rats per group; n = eight rats per group; mean ? SE. b Epinephrine plus norepinephrine; n = 10 urine samples per group. c R = Reserpine, 0.1 mg/kg, SCdaily, 16 days. dp < 0.05 (R groups compared to preceding group). e CD = Choline-deficient diet, 14 days. f CD + R = Choline-deficient diet plus reserpine. g CDS = Choline-deficient diet plus choline chloride supplement (150 mg/lO ml in tap water). * CDS + R = Choline-deficient diet plus choline chloride supplement plus reserpine.
The rats were weighed daily. The appropriate groups received subcutaneous injections of reserpine (Serpasil) beginning 2 days before the choline-deficient diet was begun and continued until the end of the experiment. Excluding the two preliminary days when all rats received Purina chow, the rats were allowed to eat and drink their respective diets ad Iibitum for 14 days. All rats were sacrificed on Day 16. Kidney and liver tissue were taken and sections were stained with hematoxylin and eosin (H & E). The kidney sections (H & E stain) were coded, and a different person graded the slides to prevent the evaluation from being prejudiced. They were graded on a scale from 0 to +4. The grading system used was modeled after that of Levensen et al. (1968). The following criteria were used for grading: 0 = no gross or microscopic pathology; +I = some hyperemia, pycnotic nuclei, and slight focal necrosis of cortex (sloughing of tubular epithelial cells); +2 = more extensive focal necrosis than +l with a few calcium deposits, a few casts, and slight subcapsular hemorrhagic areas; +3 = more extensive than +2 necrosis with moderate number of casts and larger hemorrhagic areas and more calcium deposits; +4 = extensive confluent neurotic areas, many casts extending into medullary areas, increased hemorrhage and number of calcium deposits in necrotic areas. There was no necrosis of glomeruli which would have extended grading to +5 according to Monserrat et al. (1969). Liver sections were also graded using the degree of fatty infiltration. During the period of the 16 days, five 24hr pooled urine samples of each group were obtained on Days 3, 8, 10, 15, and 16. Three milliliters of 1 N HCl were placed in the flasks that were to receive the urine samples to preserve the catecholamines prior to
SHORT
COMMUNICATION
609
assay. The urine samples were analyzed for total catecholamines (norepinephrine and epinephrine) and endogenous urinary creatinine. The catecholamines were analyzed according to the fluorescent hydroxyindole method of Udenfriend (1962). The catecholamine values were factored over the creatinine values to minimize error due to changes in filtration rate. The results of the two experiments were similar so the data were pooled. Student’s t test was used to analyze data for significance at the 5 % level (Snedecor and Cochran, 1967). RESULTS
AND
DISCUSSION
The theory that choline deficiency-induced renal necrosis is caused by an imbalance between vasoconstrictor and vasodilator substances is further substantiated by our results. Reserpine, an agent that reduces catecholamines, caused a decrease in the amount of renal necrosis (Table 1). The amount of necrosis in the CD + R rats was significantly less than that of CD rats. There was a minimal amount of necrosis in the control animals, and there was no significant difference in the degree of necrosis between control and control + R rats. There was also minimal necrosis in CDS and CDS + R. There was no difference in liver sections stained with H & E between CD and CD + R groups. Since reserpine reduced the urinary catecholamines (Table i), this would lessen the difference between the vasoconstrictor catecholamines and the decreased vasodilator acetylcholine, thus perhaps alleviating to some extent vasospasm .and ischemia. Of the total urinary catecholamines, norepinephrine was the major component (Table 1). The CD rats tended to have a higher excretion of urinary catecholamine excretion than did the control animals. In other experiments (unpublished observations) there was a significant increase in urinary catecholamines in choline-deficient rats. In the groups of rats injected with reserpine there was a significantly lower catecholamine excretion than in corresponding groups of rats without reserpine except for the CDS + R group. Although the catecholamine excretion tended to be lower in this group there was not a significant decrease compared to the CDS group with reference to norepinephrine. The correlation of decreased renal necrosis with a decrease in urinary vasoconstrictor catecholamines occasioned by reserpine lends support to the theory that the renal necrosis of choline-deficiency in rats may be due to an imbalance between vasoconstrictor catecholamines and a vasodilator such as acetylcholine. REFERENCES BAXTER, J. (1953). Protective effect of thiouracil and dibenamine against renal injury due to
choline deficiency; Influence of endocrine and autonomic systems. J. Pharmacol. Exp. 7krap. 107,394-401. GRIFFITH, W. H. AND WADE, N. J. (1939). Some effects of low choline diets. Proc. Sot. Exp. Biol. Med. 41, 188-190. LEVENSON, STANLEY M., NAGLER, ARNOLD L., GREEVER, ERVING F. AND SEIFTER, E. (1968).
Acute choline deficiency in germ free, conventionalized and open-animal-room rats: Effects of neomycin, chlortetracycline, vitamin B1z and coproghagy prevention. J. Nutr, 95,247-210.
610
SHORT
COMMUNICATION
A. J., GHOSHAL, A. K., HARTROFT, W. S. AND PORTA, E. A. (1969). Lipoperoxidation in the pathogenesis of renal necrosis in choline-deficient rats. Amer. J. Pathol. 55, 163-189. NAGLER, A. L., BAEZ, S. AND LEVENSON, S. M. (1969). Status of the microcirculation during acute choline deficiency. J. Nutr. 97,232-236. NAGLER, A. L., DETTBARN, W. D., SEIFTER, E. AND LEVENSON, S. M. (1968). Tissue levels of acetylcholine and acetylcholine esterase in weanlingrats subjected to acute choline deficiency. J. Nutr. 94, 13-19. SNEDECOR, G. W. AND C~CHRAN, W. G. (1967). Statistical Methods, 6th ed. The Iowa State University Press, Ames. UDENFRIEND, S. (1962). Tyrosine and its metabolites. In Fluorescence Assay in Biology and Medicine (N. 0. Kaplan and H. A. Scheraga, Eds.), pp. 129-160. Academic Press, New York. WILSON, R. B., HOPPE, H. AND NEWBERNE, P. M. (1972). Hemorrhagic renal necrosis in young adult rats fed a cholesterol supplemented choline deficient diet. Nut. Rep. Infer. 6,275-280. WOLBACH, S. B. AND BESSEY,0. A. (1942). Tissue change in vitamin deficiencies. Physiol. Rev. 22,233-289. J. R. BRUCE H. J. WEISE M. K. CARTER Department of Pharmacology Tulane University School of Medicine New Orleans, Louisiana Received October 7,1975; accepted February IO,1976 MONSERRAT,
