Kidney damage in rats bearing an adrenocorticotropin and prolactin secreting tumor.

The effect of chronic high plasma corticosteroids' concentration upon renal function was studied in rats bearing a transplantable pituitary mammotropic tumor which produces large quantities of ACTH and prolactin (MtTF4S). Kidney splanchnomegaly and degenerative changes of renal cortex, particularly in proximal tubules, as well as cytolysis and appearance of vacuoles were noticed in tumor bearing rats. (Na+ + K+)-ATPase activity in renal plasma membranes decreased 67% in rats with a tumor secreting ACTH and prolactin, and 64% in rats with a tumor secreting growth hormone and prolactin when compared with controls. After adrenalectomy of MtTF4S rats, kidney weight as well as plasma concentrations of urea, sodium, chloride and phosphate ions were normalized indicating the involvement of adrenal glands in the development of disturbances in renal function.

Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone.

The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3H-aldosterone-kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3H-aldosterone peak while the second 3H-aldosterone peak was eluted with Ve corresponding to Ve of free 3H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass.

Regulation of fetal Na+/K+-ATPase in rat kidney by corticosteroids.

The enzymatic differentiation of various tissues is under hormonal control in the perinatal period. Since the regulation of Na+/K+-ATPase has not been explored prenatally, the aim of this study was to determine the corticosteroid sensitivity of sodium pump maturation in the fetal period. Na+/K+-ATPase activity was both measured in kidney homogenates of fetal rats and localized by in-situ histochemistry. Sodium pump activity was first quantifiable on day 18 of fetal development as 1.4 +/- 0.17 mumol Pi/h per mg protein, and was increased 3.4-times by day 22 of gestation. While the Na+/K+-ATPase activity was the most intense in cortical tubules at an earlier fetal age (18th and 19th day), the reaction product in the medullary tubules increased with fetal age, becoming highly intense on the 21st and 22nd day of gestation. From the 18th to 21st day of fetal development homogenate Na+/K+-ATPase activity increased as a function of chronologic age. While mineralocorticoids were without any effect on Na+/K+-ATPase activity, on the last day of the fetal development, the glucocorticoid dexamethasone proved to be successful in stimulating enzyme activity in corticosteroid-suppressed animals. According to our results, glucocorticoid hormones seem to be operating as an endogenous driving force for sodium pump maturation at the end of fetal development.

Postnatal development of rat kidney plasma membrane Na-K-ATPase.

The postnatal development of the sodium transport mechanism in the rat kidney, as judged by the activity of renal plasma membrane Na-K-ATPase, was studied. Highly purified kidney plasma membranes as verified by electron microscopic and enzyme examinations, were used. Na-K-ATPase activity (mumol Pi/mg protein/h) increases exponentially from 16.9 +/- 1.94 found at birth to the mature level of 43.1 +/- 2.16 reached at the age of 41 days. This finding paralleled our results of SDS-polyacrylamide gel disc electrophoresis, showing a completely differentiated plasma membrane protein structure at birth, the maturation of which proceeds only as quantitative enrichment of some protein fractions in the further postnatal period.

A new proteinaceous factor participating in the rat ejaculate coagulation--identification, isolation and function.

A protein substance reacting with basic protein of rat seminal vesicle secretion, present in serum, testicular and epididymal homogenates and on unwashed spermatozoa, has been identified and isolated. The protein is partly of testicular origin, its concentration in male sera, testes and epididymides being age dependent and highest in sexually mature males. After castration, its concentration in male sera fails about 15%, to a comparable value present in female sera of the same age. The presence of this, as yet unrecognized, protein (molecular weight estimated at 163000) on epididymal spermatozoa is necessary for the in vitro coagulation of rat seminal vesicle proteins.

In vitro inhibition of rat kidney plasma membrane Na-K-ATPase activity by triamterene.

In vitro experiments were performed to study the effect of 2,4,7-triamino-6-phenylpteridine (triamterene), a potassium sparing diuretic agent, upon the rat kidney plasma membrane Na-K-ATPase activity. Triamterene in the concentration range from 8X10(-13) mol/l to 8X10(-3) mol/l exerted a dose-dependent inhibitory effect of the rat kidney plasma membrane Na-K-Mg-ATPase and Na-K-ATPase activities--estimated IC50 values lay at about 8X10(-3) mol/l and 8X10(-7) mol/l, respectively. The diuretic did not influence the activity of Mg-ATPase, except at very high concentration. A Lineweaver-Burk analysis demonstrated that the inhibition of Na-K-ATPase by triamterene was non-competitive. The in vitro inhibitory effect, together with an in vivo effect observed before points to a possibility that the natriuresis caused by triamterene is due, at least partly, to a direct inhibition of active sodium transport across the kidney plasma membrane.

Nephrotic changes in the rat induced by overdosage of triamterene.

Daily treatment of rats with 2,4,7-triamino-6-phenylpteridine (triamterene, Dyrenium) a potassium sparing diuretic, in daly doses of 1.5 mg, 3 mg and 4.5 mg/100 g body weight over the period of three weeks caused severe degenerative changes of renal cortical and medullary tubules resembling osmotic (sucrose) nephrosis. The undesirable side-effects were absent in the kidneys of intact rats receiving human therapeutic dose of triamterene (0.36 mg/100 g body weight) and even higher dose (1.5 mg/100 g body weight) but administered according to the prescription of treatment recommended by the manufacturer--daily over the first week and three times weekly after that. Rats .2 days after adrenalectomy administered triamterene in daily dose of 1.5 mg/100 g body weight, survived no more than 6--7 days of treatment. The results presented in this paper undoubtedly show that triamterene, although a mild diuretic agent, should be monitored in order to avoid undesirable side-effects. In addition care should be taken in patients with hypofunction of adrenal cortex.

The effect of triamterene upon the rat kidney plasma membrane Na-K-ATP-ase activity.

Triamterene (2,4,7-triamino-6-phenylpteridine) is a potassium-sparing diuretic whose mechanism of action is not clear. Experiments were performed to study the effect of triamterene upon the activity of Na-K-ATP-ase in the kidney plasma membranes. This enzyme, dissolved within the membrane bilayer, has been considered to be a biochemical vehicle for the active transport of sodium across the cell membrane. Intact and adrenalectomized rats were subjected to a five-day treatment with triamterene in a daily dose of 1.5 mg/100 g body weight. Triamterene was also administered to a group of intact, salt-loaded, rats. The activity of Na-K-ATP-ase in the kidney plasma membranes of intact and adrenalectomized rats treated with triamterene was decreased by 22.4% (p less than 0.05) and 37.2% (p less than 0.05), respectively. The activity of Na-K-ATP-ase in the renal plasma membranes of intact, salt-loaded, rats underwent greater decrease--63% (p less than 0.05). If the decreased activity of Na-K-ATP-ase in the kidney plasma membranes of rats treated with triamterene manifested the diuretic action of triamterene, results obtained in adrenalectomized rats would suggest that triamterene acts directly on the kidney, not via the adrenal glands.

Fetal rat adrenal steroidogenesis and steroid transfer to adrenalectomized mother.

On the 22nd day of gestation in rats, fetuses of acutely adrenalectomized mothers were injected subcutaneously with 0.43 muCi 4-14C-progesterone in 0.05 ml saline. Ten and 20 min after injection to fetuses, samples were taken to determine the 14C-progesterone metabolites in the plasma and adrenal glands. After extraction of the samples taken, the metabolites were separated by two-dimensional thin-layer chromatography and identified by autoradiography. 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 11beta-hydroxyprogesterone were identified in the plasma of injected fetuses, and, in far smaller amounts, in the plasma of their mothers. The plasma of noninjected fetuses also contained very small amounts of these corticoids. The fetal adrenal glands contained far smaller amounts of radioactive steroids than the fetal plasma did. The results obtained show that steroids of fetal origin can cross the placenta in and out, constituting evidence that the fetal adrenal glands are the only source of the plasma corticoids of their adrenalectomized mothers.

4-14C-progesterone conversion by the fetal rat adrenal gland in vitro.

The adrenal glands of rat fetuses with activated or inhibited pituitary adrenocorticotropic activity between the 15th and 22nd day of intrauterine development were incubated with 4-14C-progesterone for 3hr. Fetuses of intact mothers were used as controls. Conversion of progesterone into adrenal steroids was found increased on the 18th day of intrauterine development, i.e., at the time when fetal adrenocorticotropic activity begins. In comparison to controls, conversion of progesterone into fetal adrenal corticosteroids was the smallest in the fetuses of mothers with inhibited pituitary ACTH and the greatest in the adrenals of fetuses of mothers with activated pituitary adrenocorticotropic activity.

The development of sex differences in the adrenal morphology and responsiveness in stress of rats from birth to the end of life.

The appearance and development of sex difference in the adrenal cortex of rats have been studied. Morphological and secretory differences between the adrenal cortex of female and male rats begin on the 40th day of postnatal life, when females respond in stress by a greater increase in plasma corticosterone concentration. The sex difference becomes fully manifest at the age of 55 days (females have heavier absolute and relative adrenal glands and respond to stressful stimuli by a greater increase in adrenal and plasma corticosterone concentrations). At the age of 11 to 23 months the adrenal corticosterone concentrations in stress are equal in both sexes, and the absolute adrenal weights are similar from 18.5 months until death. The body weight gain is equal in both sexes during the first 50 days of life and greater in males from day 50 to 1 year of age. After this age the body weight of males remains almost the same (315 to 322 g)8 while females continue gaining weight until the end of life (196 to 231 g). This possible causal relationship between the rhythm of growth and sex difference in the adrenal glands in rats is discussed.

Steroidogenesis of the fetal adrenal gland in vitro: influence of metopirone applied in vivo and in vitro.

In vitro conversion of 4-14C-progesterone into corticosteroids in the adrenal glands of rat fetuses treated with Metopirone (Su 4885) on the last day of intrauterine development was studied. After a 1-hr incubation of the adrenal glands of fetuses injected with Metopirone, hydroxylation of progesterone into corticosterone (B), 18-hydroxycorticosterone (18-OH-B) and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) decreased and the synthesis of 11-deoxycorticosterone increased. Following preincubation of the fetal adrenal glands and 1-hr incubation with Metopirone, hydroxylation of progesterone into DOC increased and the synthesis of B decreased. Preincubation and a 2-hr incubation with Metopirone caused a decrease in the synthesis of B, 18-OH-B and 18-OH-DOC and an increase in DOC. The results constitute direct evidence of the ability of the fetal adrenal glands to synthesize all corticoids and indicate that most probably corticoids are synthesized by the fetal adrenal glands in the same way as in the adrenals of adult animals.

Inhibitory effect of prolactin on the development of fatty liver induced by ACTH in thrat.

The action and interaction of ACTH and prolactin in the development of fatty liver were investigated in intact rats treated with exogenous hormones. Administration of ACTH or of combinations of ACTH and GH to intact female rats was found to elicit significantly greater increase in liver total lipids content and concentration than administration of combinations of ACTH, or ACTH and GH, with prolactin. In addition, the results support the data reported by Bates et al. (6) that simultaneous application of GH, prolactin and ACTH reduces the effct of ACTH, upon adrenal gland weight.

Failure of the metopirone (Su4885) suppressed fetal adrenal glands to maintain corticosterone concentration of adrenalectomized pregnant rats.

Plasma corticosterone concentrations were measured following adrenalectomy of pregnant rats on the last day of gestation. Plasma corticosterone concentrations decreased 40 and 60 min after adrenalectomy by 36 and 32%, respectively, and regained the preoperative concentration 2 h following operation. When the fetuses were injected with an inhibitor of 11beta-steroid hydroxylase (Su4885) plasma corticosterone concentrations decreased 40, 60 and 120 min following adrenalectomy by 63, 67 and 71%, respectively. The results strongly suggest that the fetal adrenal glands are the source of plasma corticosterone in adrenalectomized pregnant rats.