Estela Melendez

INFLUENCE OF SEX DIFFERENCES ON THE RENAL SECRETION OF ORGANIC ANIONS

The kidney’s responsiveness to male sexual hormones has been often neglected. Renal secretion of organic anions is higher in male than in female individuals; as a consequence, most of the xenobiotics that are excreted from the organism through this pathway are eliminated more rapidly by males than by female animals. To gain further insight into this issue, we studied in vitro and in vivo characteristics of the transport of p-aminohippurate (PAH), a suitable marker for this system, in male and female rats, under different hormonal conditions. Kinetics of PAH showed a shorter elimination half-time in male than in female rats (t1/2el: male = 16.2 ± 2.1 min, female= 25.7 ± 4.5 min, P < 0.05). Castration of male rats increased t1/2el to a value similar to that of female rats (t1/2el: orchiectomized rat = 28.1 ± 7.1 min). Testosterone treatment of female rats increased the elimination rate to a value similar to that of male rats. In vitro PAH uptake by renal cortical slices from intact male rats was higher than...

Inhibitory effect of the antioxidant ethoxyquin on electron transport in the mitochondrial respiratory chain

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline, EQ) is an antioxidant used as a preservative in animal and human foods. In a previous work, we demonstrated that EQ induces an inhibition of renal secretory mechanisms that are dependent on metabolic energy; EQ inhibits renal ATPases. In the present study, we analyzed the effects of EQ on the metabolic pathways of renal and hepatic rat cells, as well as on mitochondrial and submitochondrial particles isolated from bovine heart and kidney. EQ induced a mild inhibition of oxygen uptake when it was added to whole homogenates of rat renal cortex in the presence of glucose. In contrast, a strong concentration-dependent inhibition was produced when EQ was added to preparations of intact liver mitochondria or to submitochondrial particles isolated from renal cortex. In the presence of NADH, 90% inhibition was attained at a final concentration of 1 mM EQ. The direct inhibitory effect of EQ on NADH dehydrogenase was a most relevant finding, since no inhibitor for the partial reaction of NADH-ferricyanide on this complex has been reported previously.

Inhibition of the renal uptake of p-aminohippurate and tetraethylammonium by the antioxidant ethoxyquin in the rat

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinolein, EQ) is an antioxidant used in animal foodstuffs and to prevent superficial scalding in some fruits. In renal cortical slices prepared from male rats that had consumed a diet containing EQ, EQ inhibited the specific uptake of 14C-labelled p-aminohippurate ([14C]PAH) and tetraethylammonium ([14C]TEA), markers of organic anion and cation tubular secretion, respectively. The specific uptake of [14C]TEA was five-fold more sensitive to EQ than [14C]PAH uptake (IC50 0.33 and 1.51 mM, respectively). EQ (1 mM) decreased Na+/K(+)-ATPase activity from 1.58 to 1.0 mumol inorganic phosphate/mg protein/min in renal microsomes. The activity of this enzyme provides the energy for the function of both secretory systems. These results suggest that the mechanisms by which EQ inhibits both anion and cation tubular secretion involves a decrease in the Na+/K(+)-ATPase activity. This effect leads to interference with the energy supply required for these tubular secretory mechanisms. Our results indicate that the exposure of animals or humans to high concentrations of ethoxyquin should be avoided.

Effects of eicosanoids on the water and sodium balance of the neonate

Experimental and clinical evidence support the assumption that eicosanoids affect the morphological development and the functional behaviour of the kidney during the intra-uterine and newborn periods. Inhibition of prostaglandin (PG) synthesis in the pregnant rhesus monkey resulted in renal hypoplasia in the offspring. The plasma levels of PGs are high in the newborn. Production of PGE2 by the cortical collecting duct was found to be similar in newborn and adult rabbit but the affinity of the renal tissue of the newborn for this eicosanoid was higher than that of the renal tissue of the adult rat. Based on findings in adult animals this would be expected to blunt the effect of antidiuretic hormone and account, in part, for the limited ability of the newborn to concentrate the urine. Yet, administration to unanaesthetized newborn rats of acetaminophen, a drug that inhibits the synthesis of PGE2 and thromboxane B2, blocked, rather than enhanced, the increment in urine osmolality produced by 1 h of water deprivation. The effect was absent in weaning and adult rats. A similar experimental manoeuvre increased sodium excretion in newborn but not in weaning or adult rats. Age-related differences are also evident with regard to side effects of PG synthesis inhibition. Whereas in adults adverse effects were reported only among patients with pre-existing renal disease, in newborns they occurred even after the administration of a single dose of inhibitor. Fortunately, the deleterious effects of PG synthesis inhibition on renal function appear to be reversible.

Renal handling of indomethacin and its relationship with the secretory pathway of prostaglandins

The uptake of prostaglandin E2, one of the main renal prostaglandins and of p‐aminohippurate, an indicator of the anion organic transport, by slices of kidney cortex from adult female rats was studied in the presence and in the absence of indomethacin. The drugs inhibitory effect on the uptake of prostaglandin E2 was observed both after in vivo administration as well as when it was present in the bathing media. The effect was more pronounced when the drug was given in vivo and in addition, was present in the bath. [14C]PAH uptake was inhibited by indomethacin in a dose‐related pattern and the kinetic analysis of this effect is indicative of a competitive inhibition. As expected, uptake of PAH by medullary slices was not affected by the presence of either indomethacin of PGE2. Indomethacin was more potent in inhibiting PGE2 uptake than PAH uptake.

Effects of furosemide on the renal functions of the unanesthetized newborn rat

In neonates pharmacokinetics of furosemide is very slow, as compared to adult individuals, because it is eliminated through glomerular filtration and tubular secretion, mechanisms that are not fully developed in the newborn mammal. In addition, Henles loop, the main site of action of this diuretic, is shorter in the neonate than in the adult animal. The aim of this study was to measure the response to furosemide in the unanesthetized newborn rat and to compare it with that of the adult. Furosemide (0.5, 1, 1.5, 2, 5 or 10 mg/kg body weight, i.p.) or vehicle were administered to newborn and adult rats and the effects on sodium, potassium and water balance were assessed. Despite the physiological characteristics of the neonatal kidney, furosemide induced a more marked sodium excretion and decrease in free water clearance in the newborn than in the adult rat. In contrast, urinary potassium losses elicited by furosemide were higher in the adult than in the newborn rat at similar doses. At both ages, the effects of the diuretic were dose-dependent. Plasma sodium decreased, whereas plasma potassium and hematocrit increased in the newborn rats, after the highest doses of furosemide. In the adult rat changes were less marked. Our results suggest that the neonatal kidney is more sensitive to equivalent doses of furosemide than the adult kidney.

Age-Related Differences in the Glomerular and Renal Tubular Effects of Amikacin in the Rat

Several biological responses to drugs are dependent on the age of the individual. Renal excretion of amikacin occurs mainly through glomerular filtration and this function is diminished in the young animal. We evaluated the renal response to amikacin in the unanesthetized infant rat (21-day-old) and we compared it with that of the adult rat. Either amikacin (7.5 and 75.0 mg/kg body weight, i.m.) or vehicle were administered to infant rats and the effects on sodium, potassium and water balance were assessed. Creatinine clearance (Ccr) was calculated. Amikacin induced an increment in sodium absolute excretion, whereas the Ccr decreased. In the infant rat the effects were more marked. Changes in FENa were more marked than those in Ccr, suggesting a direct effect of amikacin on the tubular reabsorption of electrolytes in addition to deleterious effects on glomerular dynamics. Our results suggest that amikacin induces more changes in renal function in the infant than in the adult rat and therefore it might be convenient to carefully evaluate the renal condition in the patients that receive this drug.

New Evidence of a Dihydropyridine-Activated Cationic Channel in the MDCK Cell Line

Newborn rat distal cells express an apical Ca2+ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca2+i in a concentration-dependent manner (IC50 = 4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca2+ channel antagonist isradipine and agonist BayK8644 increased Ca2+i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at –50 mV with perfusion solution containing 10 mM CaCl2. Nifedipine (20 µM) induced an increase in current (1.2 ± 0.3 nA), which was partially inhibited by Gd3+. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9 ± 0.8 mV (n = 21). EC50 to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.