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Prevention of Experimental Nephrosclerosis with Methyl-Testosterone
PREVENTION OF EXPERIMENTAL NEPHROSCLEROSIS WITH METHYL-TESTOSTERONE HANS SELYE
AND
E. M. ROWLEY
From the Department of Anatomy, McGill University, ivlontreal, Canada
The renotropic action of testosterone and of many testosterone derivatives has been described in various animal species and under diverse experimental conditions1-3_ It has also been shown that the renal damage produced by sublimate intoxication 4• 5 or by ligature of the ureter, with subsequent pressure atrophy 6 is inhibited by testosterone treatment. · It has been noted furthermore that nephrosclerosis and malignant hypertension are more readily produced by desoxycorticosterone acetate (D.C.A.) in females or castrated males than in intact male rats 7• 8• 9 • All these observations suggested that the testis and its hormones exert a protective action upon renal tissue. It is possible that spontaneous nephrosclerosis in man may develop owing to an endogenous over-production by a hyperactive adrenal cortex of D.C.A. or related compounds. Hence it appeared of importance to establish -whether testosterone and other testoid compounds could inhibit the nephrosclerosis experimentally produced by D.C.A. administration. We believed that if a beneficial effect could be demonstrated under such experimental conditions it might lead us to a clinically applicable therapy. Previous experiments have shown that rats may be sensitized to the nephrosclerotic action of D.C.A. by unilateral nephrectomy and by the administration of dilute NaCl solutions instead of drinking water. With this in mind we performed an experiment on 48 female albino rats weighing 57-76 g. at the onset of the experiment. These animals were subdivided into six groups each consisting of 8 rats. Groups I to III were left intact, while in Groups IV to VI the left kidney was removed at the beginning of the experiment. All groups received 1 per cent NaCl instead of drinking water. Groups I and IV, which served as intact and partially nephrectomized controls respectively, received twice daily subcutaneous injections of 5 mg. of finely ground cholesterol crystals suspended in 0.1 cc of -water. Cholesterol was given to the controls because this compound is not known to have any hormonal action yet is chemically related to the steroid hormones. Groups II, III, V and VI received twice daily subcutaneous injections of 2 mg. of D.C.A. in 0.1 cc of water. In addition to this Groups III and VI also received twice daily subcutaneous injections of 5 mg. of methylSelye, Selyc, 3 Selye, 4 Selye, 5 Selye, 6 Selye, 7 Selyc, 8 Selye, 9 Sclye, 1944. 1 2
Hans: J. Endocrinol., 1: 208, 1939. Hans: J. Urol., 42: 637, 1939. Hans: J. Urol., 46: ll0, 1941. Hans: Canad. Med. Assoc. J., 42: 173, 1940. Hans: J. Pharm. a. exper. Ther., 68: 454, 1940. Hans and Friedman, S. M.: Endocrinol., 29: 80, 1941. Hans, Hall, C. E. and Rowley, E. M.: Canad. Med. Assoc. J., 49: 88, 1943. Hans and Pentz, E. Irene: Canad. :VI. Assoc. J., 49: 264, 1943. Hans, Sylvester, Octavia, Hall, C. E. and Leblond, C. P.: J. A. ::VI. A.124: 201, 439
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FrG. 1. 1, Kidney of intact control. 2, Kidney of intact rat treated with D.C.A. Note hyaline casts, dilatation of tubules and atrophy of their lining cells. 3, Kidney of intact rat treated with D.C.A. and methyl-testosterone. Note absence of casts. The renal structure is practically normal but shows the hypertrophy of the epithelial cells characteristic of the rcnotropic effect. 4, Kidney of unilaterally nephrectomized control. Except for slight compensatory hypertrophy the renal structure is normal. 5, Kidney of unilaterally nephrectomized rat treated with D.C.A. Numerous large hyaline casts, distended tubules with atrophic lining cells and (at the base of the picture) an enlarged completely hyalinized glomerulus. 6, Kidney of unilaterally nephrectomized rat treated with D.C.A. and methyl-testosterone. Only one small hyaline cast is distinguishable at the base of the picture. The glomcruli and the epithelial cells of the tubules are well preserved, the latter showing hypertrophy due to renotropic action,
441
PREVENTION OF EXPERIMENTAL NEPHROSCLEROSIS
testosterone suspended in 0.1 cc of water. It will be noted that Groups I and IV, II and V, and III and VI respectively received the same treatment, Groups I to III differing from Groups IV to VI only in that the latter three were especially sensitized to the nephrosclerotic action by unilateral nephrectomy. All animals were lulled on the thirty-seventh day of treatment at which time their kidneys were weighed and histologically examined. Table I summarizes the average weight of the kidney as well as the degree of proteinuria and nephrosclerosis in the 6 groups. It will be kept in mind that since the animals of Groups IV and VI were unilaterally nephrectomized all weights refer to single kidneys. The histological condition of a typical average kidney representing each of the six groups is further illustrated in figures 1-6. It will be seen that both in the intact and in the unilaterally nephrectomized animals D.C.A. caused marked nephrosclerosis and increased the weight of the lcidney. This increase in weight was mainly due to the fact that after a comparatively short period of D.C.A. treatment many hyaline casts were formed and since these occluded the lumina of renal tubules they secondarily caused TABLE
1.-Effect of methyl-testosterone on nephrosclerosis elicited by D.C.A.
GROUJ? ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
I
II
I
III
Cholesterol
D.C.A.
--- ---
Weight of one kidney in g ........... N ephrosclerosis ..................... Urine protein .......................
0.629 0 0
0.976
++ +
I
V
VI
Unilaterally nephrectomized
Intact TREATMENT ............................•. , ......
IV
D.C.A. and methyltestosterone
Cholesterol
D.C.A.
--- --- --1.203 trace 0
1.005 0 0
D.C.A. and methyltestosterone ---
1.362
1.830
+++ +++
+ +
dilation of their proximal parts owing to stagnation of urine. The proteinuria (estimated with the help of the biuret reaction) was also most pronounced in the groups receiving D.C.A. alone. The renotropic action of methyl-testosterone was very obvious in all cases. It manifested itself by the increase in the renal weight of Groups III and VI as compared with Groups II and V respectively. Histologically the renotropic action was evidenced by the considerable enlargement of the epithelial cells of the nephron especially those lining the proximal and distal convoluted tubules. The most striking difference between Group II and III was the almost complete inhibition of nephrosclerosis in the latter group which received methyl-testosterone in addition to D.C.A. This beneficial effect was also evident-although perhaps not quite as dramatic-in the corresponding unilaterally nephrectomized series. Furthermore the loss of plasma protein through the urine was partly or completely inhibited in both D.C.A. treated groups by simultaneous administration of methyl-testosterone. This represents a chemical confirmation of the histologically observed absence, or at least scarcity, of hyaline, protein-like material in the lumina of the renal tubules.
442
HANS SELYE AND E. M. ROWLEY
Two additional series of experiments, planned essentially in the same manner as that described above, gave similar results; hence we shall not discuss them here in detail. On the basis of all these observations we feel justified in concluding that the renal damage caused by D.C.A. can be inhibited by methyl-testosterone. This observation confirms our view that various steroids exert two essentially different effects upon the kidney namely the nephrosclerotic and the renotropic effect. While the former is detrimental, the latter appears to be beneficial. Because of the strong "male" or testoid actions of methyl-testosterone, its clinical applicability in cases of renal damage is limited, especially in female patients. However, as we have stated in previous publications10• 11 there is good reason to believe that the renotropic and testoid effects are independent actions and although generally speaking the most renotropic steroids are also potent testoids, there are exceptions to this rule. For clinical purposes the ideal renotropic compound would be one which is completely, or at least comparatively inactive with regard to testoid potency. While such an ideal compound is as yet unknown our preliminary investigations indicate the existence of steroids . renotropic . with a more favorable t t 'd rat10 than that of methyl-testosterone12 • es 01 SUMMARY
Experiments in the rat indicate that desoxycorticosterone acetate (D.C.A.) overdosage causes nephrosclerosis, especially in animals kept on a high sodium chloride intake. Both the morphological manifestations of nephrosclerosis and the resultant proteinuria are inhibited by simultaneous treatment with methyltestosterone. In confirmation of our previous observations we find that unilaterally nephrectomized rats are particularly sensitive to the nephrosclerotic action of D.C.A., but even in these animals methyl-testosterone exerts a beneficial effect. The possible clinical implications of these observations have been discussed. ACKNOWLEDGMENTS
The expenses of this investigation were defrayed through a grant received from the Committee on Endocrinology of the United States National Research Council. The authors are also indebted to the Gelatin Products Company which furnished the steroid hormones required for this work. Selye, Hans: Rev. Canad. de Biol., 1: 577, 1942. Selye, Hans: Encyclopedia of Endocrinology, A. W. T. Franks Publishing Company, Montreal, 1943. Section I. Classified index of the steroid hormones and related compounds. 4 vols. 12 Beland, E., Masson, G. and Selye, H.: Fed. Proc. (in press). 10
11
AND
E. M. ROWLEY
From the Department of Anatomy, McGill University, ivlontreal, Canada
The renotropic action of testosterone and of many testosterone derivatives has been described in various animal species and under diverse experimental conditions1-3_ It has also been shown that the renal damage produced by sublimate intoxication 4• 5 or by ligature of the ureter, with subsequent pressure atrophy 6 is inhibited by testosterone treatment. · It has been noted furthermore that nephrosclerosis and malignant hypertension are more readily produced by desoxycorticosterone acetate (D.C.A.) in females or castrated males than in intact male rats 7• 8• 9 • All these observations suggested that the testis and its hormones exert a protective action upon renal tissue. It is possible that spontaneous nephrosclerosis in man may develop owing to an endogenous over-production by a hyperactive adrenal cortex of D.C.A. or related compounds. Hence it appeared of importance to establish -whether testosterone and other testoid compounds could inhibit the nephrosclerosis experimentally produced by D.C.A. administration. We believed that if a beneficial effect could be demonstrated under such experimental conditions it might lead us to a clinically applicable therapy. Previous experiments have shown that rats may be sensitized to the nephrosclerotic action of D.C.A. by unilateral nephrectomy and by the administration of dilute NaCl solutions instead of drinking water. With this in mind we performed an experiment on 48 female albino rats weighing 57-76 g. at the onset of the experiment. These animals were subdivided into six groups each consisting of 8 rats. Groups I to III were left intact, while in Groups IV to VI the left kidney was removed at the beginning of the experiment. All groups received 1 per cent NaCl instead of drinking water. Groups I and IV, which served as intact and partially nephrectomized controls respectively, received twice daily subcutaneous injections of 5 mg. of finely ground cholesterol crystals suspended in 0.1 cc of -water. Cholesterol was given to the controls because this compound is not known to have any hormonal action yet is chemically related to the steroid hormones. Groups II, III, V and VI received twice daily subcutaneous injections of 2 mg. of D.C.A. in 0.1 cc of water. In addition to this Groups III and VI also received twice daily subcutaneous injections of 5 mg. of methylSelye, Selyc, 3 Selye, 4 Selye, 5 Selye, 6 Selye, 7 Selyc, 8 Selye, 9 Sclye, 1944. 1 2
Hans: J. Endocrinol., 1: 208, 1939. Hans: J. Urol., 42: 637, 1939. Hans: J. Urol., 46: ll0, 1941. Hans: Canad. Med. Assoc. J., 42: 173, 1940. Hans: J. Pharm. a. exper. Ther., 68: 454, 1940. Hans and Friedman, S. M.: Endocrinol., 29: 80, 1941. Hans, Hall, C. E. and Rowley, E. M.: Canad. Med. Assoc. J., 49: 88, 1943. Hans and Pentz, E. Irene: Canad. :VI. Assoc. J., 49: 264, 1943. Hans, Sylvester, Octavia, Hall, C. E. and Leblond, C. P.: J. A. ::VI. A.124: 201, 439
H"H"0
P'.1
z>-a, a, ttj
t'
~ t,j
>ztj ttj
~ ;,:I
0
~
t,j ~
FrG. 1. 1, Kidney of intact control. 2, Kidney of intact rat treated with D.C.A. Note hyaline casts, dilatation of tubules and atrophy of their lining cells. 3, Kidney of intact rat treated with D.C.A. and methyl-testosterone. Note absence of casts. The renal structure is practically normal but shows the hypertrophy of the epithelial cells characteristic of the rcnotropic effect. 4, Kidney of unilaterally nephrectomized control. Except for slight compensatory hypertrophy the renal structure is normal. 5, Kidney of unilaterally nephrectomized rat treated with D.C.A. Numerous large hyaline casts, distended tubules with atrophic lining cells and (at the base of the picture) an enlarged completely hyalinized glomerulus. 6, Kidney of unilaterally nephrectomized rat treated with D.C.A. and methyl-testosterone. Only one small hyaline cast is distinguishable at the base of the picture. The glomcruli and the epithelial cells of the tubules are well preserved, the latter showing hypertrophy due to renotropic action,
441
PREVENTION OF EXPERIMENTAL NEPHROSCLEROSIS
testosterone suspended in 0.1 cc of water. It will be noted that Groups I and IV, II and V, and III and VI respectively received the same treatment, Groups I to III differing from Groups IV to VI only in that the latter three were especially sensitized to the nephrosclerotic action by unilateral nephrectomy. All animals were lulled on the thirty-seventh day of treatment at which time their kidneys were weighed and histologically examined. Table I summarizes the average weight of the kidney as well as the degree of proteinuria and nephrosclerosis in the 6 groups. It will be kept in mind that since the animals of Groups IV and VI were unilaterally nephrectomized all weights refer to single kidneys. The histological condition of a typical average kidney representing each of the six groups is further illustrated in figures 1-6. It will be seen that both in the intact and in the unilaterally nephrectomized animals D.C.A. caused marked nephrosclerosis and increased the weight of the lcidney. This increase in weight was mainly due to the fact that after a comparatively short period of D.C.A. treatment many hyaline casts were formed and since these occluded the lumina of renal tubules they secondarily caused TABLE
1.-Effect of methyl-testosterone on nephrosclerosis elicited by D.C.A.
GROUJ? ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
I
II
I
III
Cholesterol
D.C.A.
--- ---
Weight of one kidney in g ........... N ephrosclerosis ..................... Urine protein .......................
0.629 0 0
0.976
++ +
I
V
VI
Unilaterally nephrectomized
Intact TREATMENT ............................•. , ......
IV
D.C.A. and methyltestosterone
Cholesterol
D.C.A.
--- --- --1.203 trace 0
1.005 0 0
D.C.A. and methyltestosterone ---
1.362
1.830
+++ +++
+ +
dilation of their proximal parts owing to stagnation of urine. The proteinuria (estimated with the help of the biuret reaction) was also most pronounced in the groups receiving D.C.A. alone. The renotropic action of methyl-testosterone was very obvious in all cases. It manifested itself by the increase in the renal weight of Groups III and VI as compared with Groups II and V respectively. Histologically the renotropic action was evidenced by the considerable enlargement of the epithelial cells of the nephron especially those lining the proximal and distal convoluted tubules. The most striking difference between Group II and III was the almost complete inhibition of nephrosclerosis in the latter group which received methyl-testosterone in addition to D.C.A. This beneficial effect was also evident-although perhaps not quite as dramatic-in the corresponding unilaterally nephrectomized series. Furthermore the loss of plasma protein through the urine was partly or completely inhibited in both D.C.A. treated groups by simultaneous administration of methyl-testosterone. This represents a chemical confirmation of the histologically observed absence, or at least scarcity, of hyaline, protein-like material in the lumina of the renal tubules.
442
HANS SELYE AND E. M. ROWLEY
Two additional series of experiments, planned essentially in the same manner as that described above, gave similar results; hence we shall not discuss them here in detail. On the basis of all these observations we feel justified in concluding that the renal damage caused by D.C.A. can be inhibited by methyl-testosterone. This observation confirms our view that various steroids exert two essentially different effects upon the kidney namely the nephrosclerotic and the renotropic effect. While the former is detrimental, the latter appears to be beneficial. Because of the strong "male" or testoid actions of methyl-testosterone, its clinical applicability in cases of renal damage is limited, especially in female patients. However, as we have stated in previous publications10• 11 there is good reason to believe that the renotropic and testoid effects are independent actions and although generally speaking the most renotropic steroids are also potent testoids, there are exceptions to this rule. For clinical purposes the ideal renotropic compound would be one which is completely, or at least comparatively inactive with regard to testoid potency. While such an ideal compound is as yet unknown our preliminary investigations indicate the existence of steroids . renotropic . with a more favorable t t 'd rat10 than that of methyl-testosterone12 • es 01 SUMMARY
Experiments in the rat indicate that desoxycorticosterone acetate (D.C.A.) overdosage causes nephrosclerosis, especially in animals kept on a high sodium chloride intake. Both the morphological manifestations of nephrosclerosis and the resultant proteinuria are inhibited by simultaneous treatment with methyltestosterone. In confirmation of our previous observations we find that unilaterally nephrectomized rats are particularly sensitive to the nephrosclerotic action of D.C.A., but even in these animals methyl-testosterone exerts a beneficial effect. The possible clinical implications of these observations have been discussed. ACKNOWLEDGMENTS
The expenses of this investigation were defrayed through a grant received from the Committee on Endocrinology of the United States National Research Council. The authors are also indebted to the Gelatin Products Company which furnished the steroid hormones required for this work. Selye, Hans: Rev. Canad. de Biol., 1: 577, 1942. Selye, Hans: Encyclopedia of Endocrinology, A. W. T. Franks Publishing Company, Montreal, 1943. Section I. Classified index of the steroid hormones and related compounds. 4 vols. 12 Beland, E., Masson, G. and Selye, H.: Fed. Proc. (in press). 10
11