EFFECT OF HYPOPHYSECTOMY ON MORPHOLOGICAL APPEARANCE OF KIDNEY AND ON RENOTROPIC ACTION OF STEROID HORMONES1 HANS SELYE From the Department of Anatomy, McGill University, Montreal, Canada
In continuation of our studies on the kidney-stimulating actions of steroid hormones (Selye '39a, Martin '40), we performed a series of experiments in order to determine whether the pituitary plays any part in this "renotropic" action. Though a number of investigators have recently devoted their attention to this newly-discovered action, the numerous valuable early observations on cognate subjects have never been reviewed and hence it was felt that it would be desirable to preface the description of our own experiments by a short review of the relevant literature. Ejfect of sex on kidney structure: In fish, (Gasterosteus aculeatus L.) Titschack ('22) found that the diameter of the kidney as a whole and also of its individual segments is greater in the male than in the female especially during the breeding season. In amphibia, Sweet ('08) described close morphological relations between the network of the vasaefferentia and the kidney tubules and Spengel (1875, 1876) found that these tubules are lined by a columnar epithelium. Bidder (1846) described the canal which now bears his name and which, in Rana fusca, is connected with the dorsal collecting tubes which pass across the dorsal side of the kidney to the ureter. Thus the spermatozoa would have to pass through the kidney to the ureter. The canal is present both in males and females, although its function in the latter is not known (Knapp 1886, Ponse '27). According to Hyrtl (1863), in amphibia there are 2 types of Malpighian corpuscles, namely large ones in the ventral part and small ones in the dorsal portion of the kidney. According to Noble ('31), the latter are concerned with the sex function. Rugh ('39) found that in Rana pipiens, one may trace the course of spermatozoa through the kidney by injecting India ink into the vas efferens. The ink does not pass down through the mesial margin of the kidney to join the ureter 1 This investigation was supported by a grant from the Committee on Research in Endocrinology, National Research Council. The author is especially indebted to Drs. Gregory Stragnell and Erwin Schwenk of the Schering Corporation of Bloomfield, N . J. for the steroid hormones and to Dr. Stanley Cook of Ayerst, McKenna and Harrison Ltd. of Montreal for the A.P.L. extract used in these experiments. 110
RENOTROPIC ACTION OF HORMONES
at the posterior end; namely, where a Bidder's canal had been described, but passes into the Malpighian corpuscles and then across the kidney to the ureter (Wolffian duct). Approximately 2 per cent of the 2,000 or more Malpighian corpuscles are involved in this genital function. Regaud and Policard ('03) showed that the preterminal portion of the uriniferous tubules in Ophidia and Lacertalia shows a sex difference and therefore called it "segment sexueL" In males this segment consists of high cells which are obviously secretory, and the authors expressed the view that the products of secretion might be utilized by the spermatozoa. This sex difference was only observed in post-pubertal animals. Zarnik ('10) confirmed the existence of such a sexual segment in Ophidia but failed to observe it in Lacertalia. Reiss ('23a, b), on the other hand, demonstrated the existence of a sexual segment in Lacertalia, but emphasized that it is evident only in adult specimens and that its development is synchronous with that of the epididymis and the transformation of the spermatids into spermatozoa which takes place in males. Dornesco ('25) confirmed these results but contradicted the above theory, according to which the product of secretion is utilized by the spermatozoa, since he found that the latter develop only after the secretion is eliminated. In the guinea pig, Cirillo and Guardavaccaro ('33) noticed a sex difference in the kidney size, the organ being much larger in males than in females. In the mouse, Masui and Tamura ('26a, b) and Selye ('40b) found that the kidney of the male is significantly larger than that of the female, and we showed furthermore that the parietal lamina of Bowman's capsule and the proYimal and distal convoluted tubules are considerably higher in the male. Crabtree ('40) confirmed and extended our observations showing that in the adult male mouse, 89 per cent of the glomeruli have the "male type" of Bowman's capsule, while in mature or immature females only 13 per cent, and in immature males only 25 per cent of the Malpighian corpuscles are of this type. Iscovesco ('13d) noted that in rabbits the kidneys of males are also larger than those of females. Effect of ovariectomy on kidney structure. In the guinea pig, Cirillo and Guardavaccaro ('33) and in the mouse, Masui and Tamura ('26a, b) observed that the kidneys of ovariectomized females are only slightly smaller than those of intact females. Our material (Selye '40b) also indicates that ovariectomy causes no obvious histological change and no marked weight decrease in the mouse kidney. Several investigators (Freudenberger and Hashimoto '39, Freudenberger and Billeter '35,
Freudenberger and Howard '37) found only slight changes in the kidney weight following ovariectomy in the rat, but in most cases the spayed females had actually larger kidneys than the intact controls. On the other hand, Lauson et al. ('39), who also saw only slight deviations from the normal after ovariectomy in short term experiments, emphasized that 60 days or longer after spaying the development of the kidneys becomes subnormal. However, Hashimoto ('40) confirmed his previous statement according to which ovariectomy increases kidney growth in the rat. Korenchevsky and Ross ('40) concluded that the kidneys of ovariectomized rats show no change in size or histological structure, although their figures would indicate a small decrease in weight. Ovariectomy and Estrogen Treatment: Salvini ('36) noted that, in the rabbit, the kidney damage caused by a certain dose of uranium nitrate is much greater in ovariectomized than in normal animals while estrin exerts a protective influence on the kidneys after spaying. · In the rat, del Castillo and Sammartino ('38), Korenchevsky and Hall ('38) and Korenchevsky et al. ('39) claimed to have obtained an increase in kidney weight after administration of estradiol dipropionate, or benzoatebutyrate. Korenchevsky and Ross ('40) described "peculiar cyst-like degenerative changes in the kidneys, mostly confined to the boundary layer of the cortex and medulla," after estrogen treatment in spayed rats. Ovariectomy and Androgens: Selye ('40b) found that testosterone may increase the size of the kidney in ovariectomized mice even above that of the kidney of normal males. In the ovariectomized rat, Korenchevsky and Hall ('38), Korenchevsky et al. ('39) and Korenchevsky and Ross ('40) claimed to have observed a slight increase in kidney weight following the administration of androsterone, trans-dehydroandrosterone, testosterone propionate and androstanediol while the weaker androgens such as Li4-androstenedione and Li5-androstenediol had no definite effect (Korenchevsky and Ross '40) . Effect of ovarian hormones on the structure of the kidney in intact animals. Progesterone: Selye and Stevenson ('40) showed in mice, that progesterone causes a marked increase in the weight of the kidney both in the male and the female. This effect is antagonistic to the kidney weight depressing action of estrogens. In the rabbit, daily administration of 3-6 mg. of progesterone fails to influence the development of the kidney lesions which develop after partial occlusion of the aorta proximal to the renal arteries (Dill and Isenhour '40). Selye ('40c) observed that, in the rat, progesterone increases kidney weight and, in this respect,
RENOTROPIC ACTION OF HORMONES
acts synergistically with simultaneously given testosterone or desoxycorticosterone but antagonizes a-estradiol. Estrogens: In the mouse, Burrows and Kennaway ('34), Gardner ('35) and Selye ('39c) observed that natural estrogens often cause distention of the ureters and hydronephrosis. Similar results are obtained with diethylstilbestrol (Selye '39c). The actual weight of the kidneys decreases under the influence of continued treatment with estradiol or diethylstilbestrol, but after a certain time it returns to normal in spite of continued treatment (Selye and Stevenson '40). Pfeiffer et al. ('40) stated that estradiol dipropionate increases kidney size in mice especially when administered in conjunction with androgens, but according to our unpublished observations an increase in size following estrogens occurs only occasionally, is always transitory and merely the result of edema. In the opossum, (Didelphys virginiana) estradiol or estrone administered during the fourth to the eighteenth day of pouch life causes hydronephrosis with consequent damage to the meso- and metanephros which results in death. The condition is probably caused by obstruction due to extreme cornification of the urethra (Burns '39). Similar findings have been reported in the opossum (Trichosurus vulpecula) by Carrodus and Bolliger ('39) following treatment with commercial estrogenic preparations. In rabbits, Iscovesco ('13c) was unable to observe any change in kidney size following treatment with a lipid extract of ovaries which probably contained estrin, as judged by its other actions. Dill and Isenhour ('40) noted that estrogens do not influence the development of the kidney lesions which are usually produced in rabbits by constriction of the aorta proximal to the origin of the renal arteries. Terada ('27) was probably the first to observe degenerative changes in the kidneys of rats following treatment with follicular fluid or crude ovarian extracts. On the other hand, del Castillo and Sammartino (' ~'8) claimed that even large doses of estradiol benzoate have no effect on the kidneys of male or female rats. Selye ('40c) showed, however, that estradiol causes a pronounced decrease in kidney weight both in the male and in the female rat. This effect could, however, be prevented by testosterone, desoxycorticosterone acetate or progesterone. More recently, Korenchevsky and Ross ('40) observed cyst-like degenerative changes along the junction of cortex and medulla following treatment of intact rats with estrogenic hormones. Diethylstilbestrol causes deposition of greenish granules in the tubular epithelia which probably result from the icterus caused by this compound (Loeser '39).
Effect of testis extirpation on the kidney structure. Cirillo and Guardavaccaro ('33) noted a decrease in kidney weight after castration in guinea pigs. In the mouse, Masui and Tamura ('26a, b) and Selye ('40b) observed that the kidneys of males decrease much more markedly after gonadectomy than those of females and that in spayed animals of both sexes, the kidney weight is approximately the same. Selye ('39a, '40a, b) showed, furthermore, that the comparatively high epithelium of the parietal lamina of Bowman's capsules and of the proximal and distal convoluted tubules is a specific male sex characteristic which disappears following gonadectomy. Korenchevsky and Hall ('38), Korenchevsky, Hall and Burbank ('39), Korenchevsky, Hall, Burbank and Ross ('39) and Korenchevsky and Ross ('40) reported a decrease in kidney size following castration in rats. Testis Extirpation and Estrogens: In the rat, del Castillo and Sammartino ('38), Korenchevsky and Hall ('38), Korenchevsky, Hall and Burbank ('39) Korenchevsky, Hall, Burbank and Ross ('39), Korenchevsky, Hall and Ross ('39) observed only slight, if any, changes in kidney weight following treatment of castrate males with various estrogens; however, Korenchevsky and Ross ('40) described degenerative changes in the kidneys of animals so treated. Korenchevsky and Dennison ('34) stated that estrin enhances the kidney-enlarging effect of androgenic testis extracts in the castrate rat. Testis Extirpation and Androgens: Selye ('39a, '40a, b) showed that the characteristically male structure of Bowman's capsule and the convoluted tubules may be restored in gonadectomized mice by testosterone or its propionate. Korenchevsky and Dennison ('34) were unable to produce more than an insignificant increase in kidney weight following treatment of castrate rats with crude androgenic extracts. However, Korenchevsky and Hall ('38), Korenchevsky, Hall and Ross ('39), Korenchevsky, Hall, Burbank and Ross ('39) showed that androsterone, testosterone propionate, transdehydroandrosterone and testosterone are capable of restoring the atrophic kidney of the castrate male to or towards normal. They emphasized, however, (Korenchevsky, Hall and Ross '39) that these hormones are unable to cause an enlargement above normal. A4-androstenedione and A5-androstenediol have no definite kidney-enlarging effect under these conditions according to Korenchevsky and Dennison ('36), Korenchevsky, Hall and Ross ('39) and Korenchevsky and Hall ('39). Effect of androgens on the structure of the kidney in intact animals.
RENOTROPIC ACTION OF HORMONES
In the American chameleon (Anolis carolensis), testosterone propionate causes marked hypertrophy of the "sexual segment" in the kidney according to Noble and Greenberg ('40). In female specimens of Uromastix, Kehl ('38) obtained a similar effect with androsterone benzoate. In the mouse, Selye ('39b, '40a, b) found that treatment with large doses of testosterone or its propionate causes not only marked enlargement of the kidney as a whole beyond the size ever attained in untreated animals of either sex, but it also exaggerates the "male type" of kidney structure (high epithelium on parietal lamina of Bowman's capsule and in proximal and distal convoluted tubules) in the males, and transforms the female kidney into the male type. Hence we concluded that the testis has a normal physiological effect on the kidney. Pfeiffer et aL ('40) confirmed these findings but claimed that, although androgens enlarge the kidney, the hypertrophy is not limited to the cortical region. They found furthermore, that testosterone is considerably less active than its propionate. Iscovesco ('13) observed that a lipid extract of testis tissue enlarges the kidney in male rabbits even though the change in weight is only slight. Selye ('39a, '40c) found that, in the rat, testosterone or its propionate cause marked enlargement of the kidney above the normal level both in the female and in the male although, in the former, this enlargement is more obvious because the female kidney is originally smaller. It was emphasized, however, that this effect is not nearly as marked in rats as in mice and is not accompanied by hypertrophy of the cells in Bowman's capsule. Korenchevsky, Hall and Ross ('39) claimed at first that "The male hormones can be considered as true stimulators of certain organs in castrated rats, but not in normal animals (cf. Korenchevsky and Hall, 1939). In other words, they restore the size and weight of the liver, kidneys, spleen and heart to or towards normal, but are unable to cause supernormal enlargement." This statement is surprising in view of the fact that in the paper of Korenchevsky and Hall ('39) which they quoted, the authors expressed the view that at least a slight increase in the total weight of the kidneys and the height of the cells in the "convoluted tubules" was discernible in intact rats treated with androgens. In any case in a later publication, Korenchevsky and Ross ('40) reported evidently positive findings which were in agreement with our earlier work indicating that androgens have a distinct kidney-stimulating effect in intact rats. The authors suggested the term "nephrotrophic" to designate this action, but since the term adrenotropic action is in common usage for the adrenal stimulating effect
of hormones, we adhere to the term "renotropic" as originally proposed (Selye '40e). Selye ('40f) also showed that the renal hypertrophy caused by testosterone administration in immature rats persists for a considerable length of time after discontinuation of treatment. Ludden and Krueger ('40) confirmed our observations on the kidney-enlarging action of testosterone propionate and re-emphasized that in the rat, unlike the mouse, the enlargement of Bowman's capsule is not seen in spite of considerable kidney enlargement. They believe1 this observation to be in contradiction to our observations, but, as we said above, our earlier findings are in complete agreement with the report of these workers. It is of some interest that Paschkis et al. ('40) found that female rats treated with testosterone propionate may show "development of scrotal layers containing tubuli which apparently represent rudimentary mesonephric tubules." Artosterone, an androgenic compound extracted from the Indian summer fruit, Artocarpus integrifolia, likewise stimulates kidney growth in the rat as demonstrated by Nath and Sen Gupta ('39) . Effect of adrenalectomy on kidney structure. Marshall and Davis ('16) found that, in cats, adrenalectomy in itself has no effect on the kidneys but degenerative changes appear if such animals receive injections of diuretics. Swingle ('27) and Hartman et al. ('27) claimed however, that cloudy swelling, lipid deposition and other degenerative changes occur more or less regularly in the convoluted tubules, and to a lesser degree in Henle's loops, in the adrenalectomized cat, and Swingle holds these changes responsible for the slight albuminuria which he occasionally observed. MacMahon and Zwemer ('29) actually speak of "lipoid nephrosis" as a result of adrenal insufficiency in the cat. In the rabbit, Donati ('38) claimed to have seen degeQerative changes in the kidney following adrenalectomy. In the rat foci of degeneration are particularly frequent in the tubuli contorti of the second category according to Simpson and Korenchevsky ('35) . Silvestroni ('37) claims to have observed an enlargement of the kidneys due to hypertrophy and hyperplasia of the glomeruli in the adrenalectomized rat. On the other hand, de Wesselow and Griffiths ('38) and Gersh and Grollman ('39) were unable to demonstrate any characteristic kidney lesions in adrenalectomized rats, and the latter concluded that the positive findings of previous investigators both in this and in other species are not due to adrenal insufficiency but to operative shock. de Wesselow and Griffiths ('38) emphasized that the renal enlargement caused by thyroid hormone in the rat is even more pronounced after adrenalectomy than in intact
RENOTROPIC ACTION OF HORMONES
animals. Diaz and Levy ('39) st.ated that the renal weight in rats rendered hypertensive by partial nephrectomy is decreased in adrenalectomized animals in comparison with the controls. In such partially nephrectomized adrenalectomized rats, evidence of focal tubular damage and glomerulo-nephritis was often seen. Effect of adrenal hormones on the structure of the kidney in intact animals. Since this paper is only concerned with the renotropic effect of steroid and hypophyseal hormones, the numerous reports on the action of adrenalin on the kidney will not be discussed. Suffice it to say here that desoxycorticosterone acetate causes an increase in the size of the kidney in the rat. This is particularly marked if the hormone is given in combination with progesterone or testosterone. The adrenal cortical principle also counteracts the kidney atrophy producing effect of a-estradiol (Selye '40c). Effect of hypophysectomy on kidney structure. In the salamander, Blount ('40) showed that hypophysectomy causes a decrease in the thickness of the basement membrane in the visceral lamina of the glomerular capsule and, at the same time, a reduction in the size of the capsular space. These effects are exactly opposite to those obtained by pituitary transplants. In dogs, Lex ('30) found that the morphological changes in the kidneys during the polyuria caused by hypophysectomy are relatively inconspicuous and cannot explain the increased urine production. Dandy and Reichert ('38) emphasized that the size of the kidneys is more subnormal in hypophysectomized dogs than the body weight itself. Hypophysectomy and Partial Kidney Extirpation: Selye et al. ('34) showed that regeneration of kidney tissue is inhibited but not completely prevented in hypophysectomized rats in whom 1 kidney had been removed. They emphasized that this and other examples of regeneration in the absence of the hypophysis give definite proof that cellular growth is not necessarily dependent upon the hypophyseal growth hormone. This has been confirmed in the rat by McQueen-Williams and Thompson ('40), in the toad (Bufo arenarum) by Gonzalez ('38a, b) and in the dog by Winternitz and Waters ('40). · Hypophysectomy and Hypophyseal Extracts: Both anterior and posterior lobe extracts greatly improve the compensatory hypertrophy of the remaining kidney in unilaterally nephrectomized toads (Gonzalez '38a, b). Hypophysectomy and Thyroid Hormone: Swann ('39) showed that the kidney hypertrophy normally produced by thyroxin in the rat, far
from being inhibited by hypophysectomy, is actually more pronounced in the absence of the pituitary. The author failed to indicate either the sex or the age or even the body weight of his experimental animals and expressed the changes only by the average deviation of the weight in percentages of the normal without giving the actual kidney weights. Ejfect of hypophyseal extracts on the structure of the kidney in intact animals: Anterior Lobe Extract: In the salamander (Amblystoma punctatum), Blount ('35, '36, '40) found that implantation of several additional pituitaries into embryos results in a thickening of the basement membrane of the renal glomerulus, often accompanied by a contraction of the capillary tuft which results in an increased capsular space. He regarded this change as similar to that observed in essential hypert ension in man. In dogs, Benedict et al. ('29) and Putnam et al. ('29) observed an increase in the size of the viscera, including the kidney, following continued treatment with alkaline anterior lobe extracts. Ham and Haist ('39) claimed that treatment with a diabetogenic anterior lobe extract increases mitotic proliferation in many organs and among others in the loops of Henle after about seven daily injections. Before and after this time such a change is not discernible . Hofbauer ('32) noted marked enlargement of the kidneys in a single guinea pig which received fresh cattle anterior lobe transplants. Heinemann ('37) found that in guinea pigs treated with thyrotropic anterior lobe extract the kidneys usually show marked congestion with great dilatation of the glomerular loops. Degenerative changes were not observed.* De Donno ('38) described nephrosis-like changes, often accompanied by fatty degeneration, in the kidneys of guinea pigs treated with anterior lobe extract or luteinizing hormone (L.H.) from pregnancy urine. Iscovesco ('13b) found that a lipid extract of the anterior lobe causes kidney enlargement both in male and in female rabbits. On the other hand, Dill and Isenhour ('40) emphasized that L .H. (from pregnancy urine) in doses up to 7,200 i.u. exerts no histologically discernible effect on the rabbit kidney. Heinemann ('37) observed marked congestion of the kidneys but no degenerative changes following treatment with thyrotropic hormone in the rat. Murphy et al. ('38) found an increase in the size of the kidney after administration of potent thyrotropic extracts, and it appears probable that the reason why Hoskins ('16) was unable to obtain changes * Best and Campbell ('38) noted fat deposition in the loops of Henle and convoluted tubules accompanied by an increase in renal weight following treatment with an anterior lo be extract.
RENOTROPIC ACTION OF HORMONES
in the kidney by administration of dried pituitary powder is simply that his preparation was inactive. In sheep, Thaon ('10) found that injection of a sheep pituitary mush not only stimulates diuresis but leads to hyperemia and edema of the kidneys, often accompanied by "cellular proliferation at certain points of the glomerulus and its capsule." The tubular epithelium was usually cloudy and the change has been considered to be a sub-acute glomerulonephritis. Draper et al. ('31) noted that, in man, treatment with a pituitary extract decreased the x-ray shadow of the renal pelvis. They ascribed this to the muscle contraction-stimulating effect of their preparation. Though not strictly relevant, it is worth mentioning that Turley ('20) and Graffiin ('.39) made a wax model reconstruction of the glomerulus and proximal convoluted tubule in a case of acromegaly and found that the convoluted tubule had undergone a striking increase in length but not in diameter and that the increase in length is roughly proportional to the enlargement of the kidney as a whole. The kidney was about twice the normal size. Posterior Lobe Extracts: Milles and Hardgrove ('35) noted that, in dogs, vasopressin causes only slight changes in the normal kidney but, after denervation of the organ, it elicits mild chronic inflammatory and marked degenerative changes. In the guinea pig, Fauvet ('31b) reported thrombosis and hyaline degeneration in the kidney following the injection of Tonephin (vasopressor extract). He believed these changes to be identical with those seen in eclampsia. Ohligmacher ('33) administered sub-lethal doses of pituitrin or pitressin to guinea pigs and noted swelling of the epithelial cells often accompanied by hyaline degeneration in the convoluted tubules. Occasionally there were small hemorrhages but the glomeruli were not noticeably affected. Scheps ('34a, b) observed a persistent constriction of the small arterioles of the kidney following vasopressin administration. This was accompanied by thickening of the vascular wall. The glomeruli were congested and sometimes infiltrated by lymphocytes. The cells of the convoluted tubules showed parenchymatous, often fatty degeneration and sometimes even necrosis. Contrary to Fauvet, this author does not consider the changes to be similar to those seen in eclampsia. Vegh and Pallos ('38), vrho also noted degenerative and necrotic changes in the guinea pig kidney following posterior lobe extract administration, expressed the view that, though the changes are not unlike those seen in eclampsia, no conclusion may be drawn concerning the etiology of this disease from such experiments in which unphysiologically high doses had been given. Liu
('39) also refused to draw any conclusion regarding the pathogenesis of eclampsia from these experiments. In the rabbit, Knepper ('34) observed hemorrhages, hyaline thrombi, infarcts and albuminous degeneration of the tubule cells following treatment with posterior lobe extracts. Scheps ('34a, b) found vasoconstriction and thickening of the wall in the smaller arterial vessels of the kidney and often congestion and lymphatic infiltration of the glomeruli. The tubular epithelium showed signs of parenchymatous or fatty infiltration. Dodds et al. ('34) observed "signs of a toxic effect" in the kidneys of rabbits treated with a posterior lobe extract. Later Liu and Noble ('38) showed that direct injection of vasopressin into the kidney arteries causes infarction and histological signs of glomerular and tubular damage while oxytocic preparations had no such effect. Dill and Isenhour ('40) produced definite kidney lesions in rabbits receiving 60-120 pressor units of vasopressin. Byrom ('37) produced ischemic, degenerative changes and infarcts in the kidneys with large doses of vasopressin in the rat. He regarded these lesions as the result of renal vasoconstriction. Fauvet ('31a) noted thickening of the glomerular capsule often accompanied by swelling or hyaline degeneration of the convoluted tubules in rats receiving posterior lobe extract. Hypophyseal Extracts and Estrogens : Byrom ('38) noted that, in the rat, vasopressin in doses which normally cause only slight or no renal damage elicits marked infarction in the kidneys of estradiol benzoate pretreated females. From the above survey, we may conclude that a sex difference in the structure of the kidneys has definitely been established, not only in lower vertebrates but also in the mammals. Furthermore we may say that the kidneys of males are usually larger than those of females and that extirpation of the testes reduces kidney size while androgen therapy restores it to, or above, normal in castrates of both sexes. Steroids of the corpus luteum and the adrenal cortex also cause kidney enlargement but, in this respect, they are less active than the androgens. Hypophysectomy decreases the size of the kidneys and inhibits, but does not prevent the compensatory hypertrophy which normally occurs following partial nephrectomy. With this in mind, it seemed important to establish whether the renotropic action of the steroid hormones is direct or whether it is mediated by the hypophysis. If the latter were true, hypophysectomy should completely prevent the renotropic action of steroids just as it prevents the mammogenic action of these compounds which has thus been proven to be an indirect one (Selye '40d).
RENOTROPIC ACTION OF HORMONES
Experimental. In our first experimental series, we used 24 female albino rats weighing approximately 90 gm. on the average. These were divided into 4 groups of 6. The animals in group 1 served as untreated intact controls, group 2 was treated with 10 mg. of progesterone administered daily subcutaneously in 0.4 cc of peanut oil. Group 3 consisted of hypophysectomized controls and group 4 was hypophysectomized and treated with 10 mg. of progesterone daily in the same manner as group 2. The hypophysectomies in this and in all subsequent experiments were performed by the parapharyngeal approach. Injections were begun on the day of hypophysectomy and the controls, both hypophysectomized and intact, received the same amount of peanut oil as was given to the progesterone-treated animals as a solvent for the hormone. Treatment was continued for 20 days so that each animal received a total of 200 mg. of progesterone. On the twenty-first day, all rats were killed and their kidneys weighed and histologically examined. Table 1 summarizes our results giving the average organ and body weights of each group in TABLE
1.-Ejfect of progesterone on kidney of intact and hypophysectomized female rat TREATMENT
Initial body weight ........... ... ' Weight gain .............. ....... Kidney weight ....
106 +42 1.212
107 +43 1.254
grams. The kidneys of the hypophysectomized untreated animals are considerably smaller than those of the intact controls and this decrease is more pronounced than could be expected from the fact that the hypophysectomized animals did not gain in body weight after the operation while the intact controls gained about 40 per cent. The experiments to be described subsequently in this communication confirm the fact that, in the hypophysectomized rat, kidney development is subnormal even when expressed in percentage of the total body weight. On the other hand, the table indicates that, unlike our previous experiments in the mouse, the kidney of the rat is not very sensitive to the renotropic action of progesterone since the increase in the average weight of the organ was only insignificant both in the intact and in the hypophysectomized group. Our subsequent experiments in which desoxycorticosterone acetate and androgens were used will show that comparatively speaking, the renotropic action of the latter is more pronounced than that of progesterone in the rat.
In our second experiment, we investigated the renotropic action of desoxycorticosterone acetate. The experiment was performed in exactly the same manner as the previously reported series in which progesterone was given, the only difference being that desoxycorticosterone acetate was administered in 4 mg. daily doses, this amount being dissolved in 0.2 cc of peanut oil. Table 2, in which our findings are summarized, indicates clearly that this compound stimulates kidney growth both in the intact and in the hypophysectomized animal, and this increase proved to be statistically significant in both cases. Yet even these comparatively large doses of desoxycorticosterone acetate failed to prevent the inhibition of kidney development caused by hypophysectomy. This is noteworthy because in the dose given, desoxycorticosterone acetate causes compensatory atrophy of the adrenal cortex which may be taken as an indication that this amount given daily exceeds the normal cortical hormone production of the rat. Since the daily administration TABLE
2.-Ejfect of D.C.A .* on kidney of intact and hypophysectomized female rat
Initial body weight . . . . . .......... Weight gain .. ... .. . . ... . .... .. . . Kidney weight . . . ... ... . . . . ......
99 0 0.726
HYPO PHY SECTOMIZED D.C.A,
105 -5 0.818
* Desoxycorticosterone acetate. of 10 mg. of progesterone in the previous series caused marked mammary gland proliferation and uterus enlargement, such as are only seen during pregnancy, it may also be concluded that corpus luteum hormone production at least outside of pregnancy never equals this amount. We are stressing these facts because they indicate that the renal atrophy caused by ablation of the pituitary cannot merely be ascribed to the resulting decrease in the production of these ovarian and adrenal cortical steroids. Even though the latter exert some direct renotropic effects in the absence of the pituitary, it appears that the pituitary must have other means of stimulating the kidney besides its luteinizing and adrenotropic effects. In order to obtain further evidence supporting the view that steroid hormones may exert a direct effect on the kidney, a third series of experiments was performed in which intact and hypophysectomized rats were treated with daily doses of 4 mg. of testosterone for 20 days, the autopsy being performed on the twenty-first day just as in the previous
RENOTROPIC ACTION OF HORMONES
series. Table 3 indicates that this dose of the male hormone likewise caused a kidney enlargement both in the intact and the hypophysec1:omized animals. It should be emphasized here again that this amount of testosterone must certainly be regarded as being in excess of the normal daily male hormone production, as it caused enormous enlargement of the uterus, vagina and preputial glands in this series and produced comparable enlargement of the male accessory glands as shown by other experiments not reported here in detail. Since even testosterone, which among the steroid hormones so far investigated proved to have the most pronounced renotropic activity, failed to restore the atrophic kidney of the hypophysectqmized animal to normal, it appeared of interest to determine whether the androgens secreted by the testis under natural conditions would be more efficient in this respect. In order to determine this, relatively pure preparations of follicle stimulating hormone (F.S.H.) and luteinizing hormone (L.H.) were TABLE
3.-Ejfect of testosterone on kidney of intact and hypophysectomized female rat TllEATllENT
Initial body weight ............... Weight gain ..................... Kidney weight ...................
98 +s6 1.562
99 +48 1.618
HYPOPHYSEC• TOHIZED TESTOSTERONE
101 -11 0.822
tested both alone and in combination with testosterone on hypophysectomized rats. For this purpose a series of 7 groups, each consisting of 6 male albino rats were treated as follows: Group 1, intact controls treated with oil; group 2, hypophysectomized controls treated with oil; group 3, hypophysectomized treated with L.H.; group 4, hypophysectomized treated with L.H. and testosterone; group 5, hypophysectomized treated with testosterone; group 6, hypophysectomized treated with F.S.H.; group 7, hypophysectomized treated with F.S.H. and testosterone. "A.P.L.," a purified pregnancy urine preparation made by Ayerst, McKenna and Harrison, Montreal was used as a source of L.H. and "Antex," a pregnant mare serum preparation made by L¢vens Kemiske Fabrik, Copenhagen was employed as a source of F.S.H. The former was administered in doses of 100, the latter in doses of SO i.u. in aqueous solution subcutaneously once daily. Testosterone was given once daily in doses of 10 mg. in 0.4 cc of peanut oil by the subcutaneous route. Unlike the previous series in which injections were begun im-
mediately after ablation of the pituitary in this group, a period of 14 days was allowed between the hypophysectomy and the initiation of the treatment in order to allow severe testis and kidney involution. The animals were then treated for 14 days and sacrificed on the twentyninth day after hypophysectomy. In the course of the experiment, three animals in group 4 and two in group 7 succumbed. The average organ weights given in table 4 are based only on the remaining animals which survived throughout the experiment. It is noteworthy that all casualities occurred in groups receiving gonadotropic hormones in conjunction with testosterone. The figures in table 4 clearly indicate that even in combination with L.H. or F.S.H., testosterone fails to restore the kidney to the normal TABLE
4.-Ejfect of testosterone and gonadotropic extracts on kidney of intact and hypophysectomized male rats BYPOPRY·
RYPOPRY· RYPOPRY· SECT· SECT· OMIZED
L.B. AND TESTOS-
RYPOPRY· RYPOPRY· SECTOMIZED TESTOS·
HYPOPHY• SECTOHIZED F,S,B.
SECT· OMIZED F.S,H. AND
- - - - --- - -
Initial body weight ...... . . . 75 92 84 89 78 91 90 Weight gain ......... . ...... +103 -1 -7 -4 -1 +4 +17 Kidney .... . .. ...... ... .... 1. 755 0 .737 0.892 0 .973 0.902 0 .932 0.881 Testis ... ..... .. . . . . . . . . .. . 2.257 0.164 0.532 0.607 0.394 1.040 0.960 Seminal ves. . ... . . . ..... . .. 0.339 0.009 0.320 0.467 0.552 0.441 0.515 0.503 0.052 0.371 0.385 0.441 Prostate ... . ... ... . . . ... .. . 0.494 0.465 Epididymis .. .. . .. ......... 0.412 0.036 0.172 0.226 0.208 0 . 194 0.170
size but both these gonadotropic hormones have some renotropic action by themselves, probably because they stimulate the testis to produce androgens. The fact that, in all the treated hypophysectomized animals, the testes were stimulated by the treatment, and the kidneys were also enlarged in comparison with the untreated hypophysectomized controls, emphasized the close relationship between gonadal and kidney development. In accordance with previous observations on this subject, we find that the testes of hypophysectomized testosterone-treated animals are larger than those of untreated hypophysectomized controls. This relative increase in testis size is not as marked as in the groups receiving gonadotropic hormones, yet the kidneys are greatly enlarged under the influence of testosterone alone. This appears to support the view that the renotropic action of this androgen is direct while that of
RENOTROPIC ACTION OF HORMONES
the gonadotropic hormones is mediated by the testis. The table shows furthermore, that the seminal vesicles, prostates and epididymes are much more markedly stimulated by testosterone in the hypophysectomized rat than the kidneys. The seminal vesicles of all treated hypophysectomized groups have been restored by the various hormones to or even above the level observed in the not hypophysectomized controls while the kidneys even in the group in which development was optimal were not much larger than half the normal size. Discussion. The observations recorded in this communication clearly indicate that the steroid hormones exert a certain renotropic action even in hypophysectomized animals but doses of progesterone, testosterone or desoxycorticosterone, which are certainly in excess of the amount normally produced in the organism, failed to restore the kidney to its normal size. From this, it may be concluded that, unlike the uterus or the seminal vesicles which are completely maintained and may even be stimulated above the normal size by treatment with steroid hormones, the maintenance of the kidney is dependent on substances other than the steroids used in these experiments. It is also noteworthy in this connection that our previous observations showed that the renal atrophy caused by gonadectomy in males is less severe than that elicited by hypophysectomy and may be com:eletely prevented by suitable androgen therapy. It is clear, therefore, that besides the stimulation of testosterone, progesterone and desoxycorticosterone production by the endocrines which are under the influence of the tropic pituitary hormones, the hypophysis must exert some other kidney growth-stimulating action. Several possibilities must be considered. It is conceivable that the thyrotropic hormone which stimulates thyroid hormone production plays an important role in the renotropic effect of the pituitary as it has been shown that thyroid hormone also causes marked kidney enlargement. It is also possible, and in fact very probable, that the growth hormone which stimulates the development of all organs is involved in this effect. Hence there is no reason to believe without further proof, that the pituitary produces a special renotropic hormone different from the known pituitary principles, ahhough this possibility cannot be excluded. The most probable explanation of our observations would be to assume that various pituitary hormones co-operate in order to obtain optimal kidney development. Among these hormones, the corticotropic, gonadotropic, thyrotropic and the growth hormones must be considered. Since renotropic stimulation by steroids is also dependent on the anterior lobe which
stimulates the production of these steroids by the other endocrines, the important role of the hypophysis in the maintenance of the normal kidney structure appears to be well established. We did not discuss the histological changes observed in the kidney after the administration of the steroid hormones since this has been done in previous communications mentioned in the introduction to this paper. Suffice it to re-emphasize that it is mainly the tubular apparatus which enlarges as a result of treatment with these compounds and that it is involution of the tubular apparatus which accounts for most of theatrophy observed after hypophysectomy. As an incidental observation, it may be worth mentioning that the pancreas of normal and hypophysectomized animals treated with desoxycorticosterone, progesterone, and testosterone was also markedly enlarged in the experiments, but, since this is not relevant to the main problem of this paper, we did not include the full description of these findings in this communication. SUMMARY AND CONCLUSIONS
Progesterone, desoxycorticosterone and testosterone exert a definite renotropic action both in the intact and in the hypophysectomized rat. Their activity in the absence of the pituitary shows that the kidneystimulating effect of these hormones is not mediated by the hypophysis but is probably direct. While previous observations showed that moderate doses of the above steroids suffice to prevent the slight kidney involution normally seen after castration, the experiments reported in this communication indicate that, even in doses which exceed the amounts normally produced in the body, progesterone, desoxycorticosterone and testosterone fail to restore the markedly atrophic kidney of the hypophysectomized rat to normal. It is concluded that under physiological conditions besides the gonadotropic and corticotropic hormones, the pituitary possesses additional means for ' the maintenance of the normal kidney structure. Follicle-stimulating and luteinizing preparations exert a definite renotropic action in the non-castrate hypophysectomized male rat, probably because they stimulate androgen production by the testes. However these gonadotropic hormones are unable to restore the kidney size to normal even if they are given in conjunction with large doses of testosterone. Since thyroid hormone likewise stimulates kidney growth, it appears
RENOTROPIC ACTION OF HORMONES
probable that the thyrotropic hormone is also responsible for part of the renotropic effect of the hypophysis. The growth hormone which conditions the growth of the body as a whole must similarly be considered in this connection. The experimental evidence now at hand does not necessitate the assumption of a special renotropic pituitary hormone, although deficiency in the gonadotropic and adrenotropic hormones, which are responsible for the production of the kidney-stimulating steroids, does not suffice to explain the pronounced renal atrophy caused by hypophysectomy. It appears most probable that the kidney is dependent for the maintenance of its normal structure on the balanced production of the growth and thyrotropic principles in addition to the gonadotropic and adrenotropic hormones. REFERENCES BENEDICT, EDWARD B., PUTNAM, T. J., AND TEEL, H. M.: Early changes produced in dogs by the injections of a sterile active extract from the anterior lobe of the hypophysis. Am. J. Med. Sci., 179: 489, 1930. BEST, C.H. AND CAMPBELL, JAMEs: The effect of anterior pituitary extracts on the liver fat of various animals. J. Physiol., 92: 91, 1938. BIDDER, F. H.: Vergleichend-anatomische und histologische Untersuchungen tiber die mii.nnlichen Geschlechts- und Hamwerkzeuge der nackten Amphibien, 1846. BLOUNT, RAYMOND F.: Kidney glomerulus of hypertension produced experimentally by pituitary excess. Proc. Soc. exper. Biol. & Med., 32: 650, 1935. - - - : The glomerular basement membrane of hypertension in experimentally produced hyperpituitaris. Anat. Rec., 65: 1, 1936. - - - : Quantitative alteration in renal corpuscles of salamanders through variation in hypophyseal tissue. Proc. Soc. exper. Biol. & Med., 43: 537, 1940. BuRNs, R. K., JR.: Effects of female sex hormones in young opossums. Proc. Soc. exper. Biol. & Med., 41: 270, 1939. BURROWS, HAROLD, AND KENNAWAY, N. M.: On some effects produced by applying oestrin to the skin of mice. Amer. J. Can., 20: 48, 1934. BYROM, F. B.: Morbid effects of vasopressin on the organs and vessels of rats. J. Path., 45: 1, 1937. - - - : The effect of oestrogenic and other sex hormones on the response of the rat to vasopressin. Lancet, 1: 129, 1938. CARRODUS, ARTHUR, AND BOLLIGER, ADOLPH: The effect of oestrogenic hormone on prostate of marsupial Trichosurus vulpecula. Med. J. Australia, 2: 633, 1939. DEL CASTILLO, E. B., AND SAMMARTINO, R.: Action des fortes doses prolongees d'oestrone sur les organes endocrines et sexuels du rat. C. r. Soc. Biol., 129: 870, 1938. CrRrLLO, N., AND GuARDAVACCARO, G.: II peso dei reni, dei surreni, delle vescicole seminale e della milza in seguito a gonadectomia in Cavia cobaya. Scritti biologici, 8: 253, 1934. CRABTREE, CHARLOTTE: Sex differences in the structure of Bowman's capsule in the mouse. Science, 91: 299, 1940. DANDY, WALTER E., AND REICHERT, F. L.: Studies on experimental hypophysectomy in dogs. III. Somatic, mental and glandular effects. Bull. Hopkins Hosp., 62: 122, 1938. DIAZ, J. T., AND LEVY, SANFORD E.: Studies on experimental hypertension in the rat. Am. J. Physiol., 125: 586, 1939. DILL, L. V., AND ISENHOUR, C. E.: An attempt to produce increased susceptibility to renal ischemia in rabbits by means of endocrine preparations. Endocrinol., 26: 863, 1940.
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