Studies on the renal tubular secretion of organic acids in the dog

의학과/박사[한글] Studies on the Renal Tubular Secretion of Organic Acids in the Dog Kyu Chul Cho Department of Pharmacology and Toxicology Yonsei University College of Medicine, Seoul, Korea (Directed by Prof. Woo Cheo Lee) Since Marshall and Vickers (1923) succeeded in demonstrating the fact that phenol red is secreted by the renal tubule, many substances have been shown to be excreted by the kideny in a manner similar to phenol red. It is generally accepted at the present time that certain weak organic acids such as phenol red, para-aminohippuric acid (PAH) and Diodrast are actively secreted by the renal tubule through a common transport system (Smith and Smith, 1938). When the renal tubule was subjected to more than one of these substances, the tubular secretion of each substance was found to be inhibited in a competitive manner. The underlying mechanism for this competition is not clear. It has been shown that Diodrast is a more powerful competitive inhibitor than PAH when these two compounds were administered (Josephson et al., 19i2; Josephson et al., 1953; Forster and Hong, 1959). Furthermore, evidence that the internal secretions of endocrine organs such as hypophyseal, adrenocortical and thyroid glands exert powerful renotropic action especially on the tubular functions has been accumulated (Smith, 1951). White et al. (1940, 1941, 1947, 1949) reported numerous experimental findings concerning tubular functions associated with anterior hypophysis and concluded that the renotropic effect of anterior hypophysis is attributable to growth hormone, but it was also observed that the growth hormone exert only a minor effect on the restorations of the kidney function, when the animals were subjected to the adrenalectomy beforehand. Reductions in renal plasma flow, glomerular filtration rate and tubular secretory mass following the adrenalectomy were observed in the dog (Harrison and Barrow, 1937; Gaudino and Levitt, 1949), and the rapid alleviation of altered functions by means of salt administration but not with adrenal cortical extract was also reported (Harrison and Darrow, 1937). But Friedman et at.(1948) and Roemmelt et al. (1949) established that the administration of a sufficient amount of adrenal cortical extract following adrenalectomy resulted in complete restoration of tubular function to normal state, while Kochakian (1944) observed an acceleration of growth rate of renal tubules by the administrations of steroid hormones. Recently, Farah et at. (1956) observed reduction of PAH uptake in the renal slices of hypophysectomized rat, as well as the restoration to the normal state followed by the administration of growth hormone and thyroid hormone. However, insignificant effects were obtained with the administrations of ACTH or cortisone. It seems clear that the hypophysis and the adrenal cortex exert some effects on the renal tubular function, but the results obtained by many investigators are confusing and, moreover, the studies on the effects of these hormone as administered by a rapid and constant infusion are scarce. The purpose of the present investigation is to investigate the following two aspects : a quantitative analysis on the kinetic aspect of competitive inhibition between PAH and Diodrast, and a clarification of the effect of steroid hormones on the renal tubular transport. Materials and Meathods Experiments were carried out in 45 anesthetized female mongrel dogs. After anesthesia with pentobarbital sodium (30mg/kg), a trachea was intubated, and cannulas were introduced into the femoral artery and vein for the purposes of blood sampling and infusion. The urinary bladder was ,catheterized for quantitative collections of urine. When the animal recovered from the effect of surgery, a control experiment was undertaken. In experiment A: Priming doses of inulin (50mg/kg) and of a substrate (120mg/kg of PAH or 80mg/kg of Diodrast) were infused, following which a constant infusion of inulin(1.0mg/kg/min) was started. The inulin was dissolved in 0.9% Nacl solution containing 2M urea in order to maintain a good urine flow. After a 20 minute equilibration period, blood and urine samples were taken at 10 minute intervals for a period of an hour. At the time of each urine collection, the urinary bladder was washed with an aliquot of distilled water once or twice, depending upon the rate of urine flow. Upon completing the control experiment, the identical procedure was repeated in the presence of a competitive inhibitor. Diodrast was employed as the competitive inhibitor when the PAH secretion was studied in the experiment, and vice versa. While the constant infusion of inulin was being maintained, the original substrate in the same amount as in the control experiment was administered along with the corresponding competitive inhibitor. The priming dose of PAH or Diodrast, when used as a competitive inhibitor, was 120 or 80mg/kg, respectively. Immediately following the administration of this priming dose, Diodrast or PAH as a competitive inhibitor was infused contineously at the rate of 2.76 or 2.03mg/kg/min, respectively, throughout the period of competition studies. In experiment B: Similar procedure as in experiment (A) was carried out, except that the constant infusion of PAH or Diodrast as a substrate was maintained at the rate of 2.8 or 2.03mg/kg/min, respectively, that ACTH, cortisone, desoxycorticosterone, testosterone, estrogen and progesterone were infused instead of competitive inhibitor in the experimental study. Both plasma and urine samples were analyzed for inulin (Shreiner, 1950), PAH (Smith et al., 1945) and Diodrast (Alpert, 1941). The computations of the various kinetic constants were based on Michaelis-Menten equations as described by Neilands and Stumpf(1958) in experiment(A). Results Experiment A: The manner in which PAH and Diodrast are secreted competitively by the renal tubules has been studied in anesthetized dogs, with special emphasis on the kinetic aspect, and the following results were obtained. 1. The transfer maxima of PAH and of Diodrast were 5.0 and 1.6μM/min/kg, respectively. 2. The values of Km for the PAH and Diodrast transport system were 0.223 and 0.15 μM/ml, respectively. 3. The dissociation constants of the carrier-PAH and carrier-Diodrast complex were 0.145 and 0.0335μM/ml, indicating that Diodrast is 4 times more strongly bound to the carrier than PAH is. Experiment B: The effects of various steroid hormones on the renal tubular transport system with special emphasis on the transport of PAH and Diodrast has been studied, and the following results were obtained. 1. Constant infusion of ACTH at the rate of 0.05u/kg/min induced marked elevations of Tm PAH and Tm D while urine flow, glomerular filtration rate and plasma concentration of PAH or Diodrast were maintained at a constant level throughout the experimental study. 2. Administrations of cortisone acetate(0.5mg/kg/min) and desoxycorticosterone gluconate (0.4mg/kg/min) resulted in significant increases in Tm values of PAH and Diodrast to a similar extent. 3. In the comparative study of the effects of testosterone propionate (0.16mg/kg/min), estrogen (130u/kg/min) and progesterone (0.4 mg/kg/min), there were no significant changes in the Tm PAH values in estrogen and progesterone group, but a marked elevation of Tm PAH in testosterone group in either female or male dogs was observed. The changes in Tm PAH in testosterone group seem to be due to the anabolic effects of testosterone. [영문] Since Marshall and Vickers (1923) succeeded in demonstrating the fact that phenol red is secreted by the renal tubule, many substances have been shown to be excreted by the kideny in a manner similar to phenol red. It is generally accepted at the present time that certain weak organic acids such as phenol red, para-aminohippuric acid (PAH) and Diodrast are actively secreted by the renal tubule through a common transport system (Smith and Smith, 1938). When the renal tubule was subjected to more than one of these substances, the tubular secretion of each substance was found to be inhibited in a competitive manner. The underlying mechanism for this competition is not clear. It has been shown that Diodrast is a more powerful competitive inhibitor than PAH when these two compounds were administered (Josephson et al., 19i2; Josephson et al., 1953; Forster and Hong, 1959). Furthermore, evidence that the internal secretions of endocrine organs such as hypophyseal, adrenocortical and thyroid glands exert powerful renotropic action especially on the tubular functions has been accumulated (Smith, 1951). White et al. (1940, 1941, 1947, 1949) reported numerous experimental findings concerning tubular functions associated with anterior hypophysis and concluded that the renotropic effect of anterior hypophysis is attributable to growth hormone, but it was also observed that the growth hormone exert only a minor effect on the restorations of the kidney function, when the animals were subjected to the adrenalectomy beforehand. Reductions in renal plasma flow, glomerular filtration rate and tubular secretory mass following the adrenalectomy were observed in the dog (Harrison and Barrow, 1937; Gaudino and Levitt, 1949), and the rapid alleviation of altered functions by means of salt administration but not with adrenal cortical extract was also reported (Harrison and Darrow, 1937). But Friedman et at.(1948) and Roemmelt et al. (1949) established that the administration of a sufficient amount of adrenal cortical extract following adrenalectomy resulted in complete restoration of tubular function to normal state, while Kochakian (1944) observed an acceleration of growth rate of renal tubules by the administrations of steroid hormones. Recently, Farah et at. (1956) observed reduction of PAH uptake in the renal slices of hypophysectomized rat, as well as the restoration to the normal state followed by the administration of growth hormone and thyroid hormone. However, insignificant effects were obtained with the administrations of ACTH or cortisone. It seems clear that the hypophysis and the adrenal cortex exert some effects on the renal tubular function, but the results obtained by many investigators are confusing and, moreover, the studies on the effects of these hormone as administered by a rapid and constant infusion are scarce. The purpose of the present investigation is to investigate the following two aspects : a quantitative analysis on the kinetic aspect of competitive inhibition between PAH and Diodrast, and a clarification of the effect of steroid hormones on the renal tubular transport. Materials and Meathods Experiments were carried out in 45 anesthetized female mongrel dogs. After anesthesia with pentobarbital sodium (30mg/kg), a trachea was intubated, and cannulas were introduced into the femoral artery and vein for the purposes of blood sampling and infusion. The urinary bladder was ,catheterized for quantitative collections of urine. When the animal recovered from the effect of surgery, a control experiment was undertaken. In experiment A: Priming doses of inulin (50mg/kg) and of a substrate (120mg/kg of PAH or 80mg/kg of Diodrast) were infused, following which a constant infusion of inulin(1.0mg/kg/min) was started. The inulin was dissolved in 0.9% Nacl solution containing 2M urea in order to maintain a good urine flow. After a 20 minute equilibration period, blood and urine samples were taken at 10 minute intervals for a period of an hour. At the time of each urine collection, the urinary bladder was washed with an aliquot of distilled water once or twice, depending upon the rate of urine flow. Upon completing the control experiment, the identical procedure was repeated in the presence of a competitive inhibitor. Diodrast was employed as the competitive inhibitor when the PAH secretion was studied in the experiment, and vice versa. While the constant infusion of inulin was being maintained, the original substrate in the same amount as in the control experiment was administered along with the corresponding competitive inhibitor. The priming dose of PAH or Diodrast, when used as a competitive inhibitor, was 120 or 80mg/kg, respectively. Immediately following the administration of this priming dose, Diodrast or PAH as a competitive inhibitor was infused contineously at the rate of 2.76 or 2.03mg/kg/min, respectively, throughout the period of competition studies. In experiment B: Similar procedure as in experiment (A) was carried out, except that the constant infusion of PAH or Diodrast as a substrate was maintained at the rate of 2.8 or 2.03mg/kg/min, respectively, that ACTH, cortisone, desoxycorticosterone, testosterone, estrogen and progesterone were infused instead of competitive inhibitor in the experimental study. Both plasma and urine samples were analyzed for inulin (Shreiner, 1950), PAH (Smith et al., 1945) and Diodrast (Alpert, 1941). The computations of the various kinetic constants were based on Michaelis-Menten equations as described by Neilands and Stumpf(1958) in experiment(A). Results Experiment A: The manner in which PAH and Diodrast are secreted competitively by the renal tubules has been studied in anesthetized dogs, with special emphasis on the kinetic aspect, and the following results were obtained. 1. The transfer maxima of PAH and of Diodrast were 5.0 and 1.6μM/min/kg, respectively. 2. The values of Km for the PAH and Diodrast transport system were 0.223 and 0.15 μM/ml, respectively. 3. The dissociation constants of the carrier-PAH and carrier-Diodrast complex were 0.145 and 0.0335μM/ml, indicating that Diodrast is 4 times more strongly bound to the carrier than PAH is. Experiment B: The effects of various steroid hormones on the renal tubular transport system with special emphasis on the transport of PAH and Diodrast has been studied, and the following results were obtained. 1. Constant infusion of ACTH at the rate of 0.05u/kg/min induced marked elevations of Tm PAH and Tm D while urine flow, glomerular filtration rate and plasma concentration of PAH or Diodrast were maintained at a constant level throughout the experimental study. 2. Administrations of cortisone acetate(0.5mg/kg/min) and desoxycorticosterone gluconate (0.4mg/kg/min) resulted in significant increases in Tm values of PAH and Diodrast to a similar extent. 3. In the comparative study of the effects of testosterone propionate (0.16mg/kg/min), estrogen (130u/kg/min) and progesterone (0.4 mg/kg/min), there were no significant changes in the Tm PAH values in estrogen and progesterone group, but a marked elevation of Tm PAH in testosterone group in either female or male dogs was observed. The changes in Tm PAH in testosterone group seem to be due to the anabolic effects of testosterone.restrictio