Non-metallothionein-bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat.

Male rats were injected SC with 0.6 mg Cd/kg/day for 5 days per week for 2, 4, 6, and 8 weeks. Liver and kidney were examined morphologically and analyzed for metallothionein, cadmium, zinc, and copper. Morphologic changes were found in kidney but not in liver. The earliest ultrastructural change consisted of myelin figures in vacuoles in cytoplasm of proximal tubular lining cells reflecting degeneration of membranes. This change occurred after 4 weeks with 801 +/- 25 nmol/g (89.9 micrograms/g) total kidney cadmium or 390 nmol/g (43.7 micrograms/g) of cadmium not bound to metallothionein. Similar changes were observed after 6 weeks but after 8 weeks pathological changes consisted of focal cellular necrosis and interstitial fibrosis. Other ultrastructural changes included altered mitochondria and increased numbers of microbodies. Renal cadmium after 8 weeks exposure was 1827 +/- 48 nmol/g (215.3 +/- 5.8 micrograms/g) or 628 nmol/g (70.2 micrograms/g) of cadmium not bound to metallothionein. Total cadmium was higher in liver than in kidney but partitioning between bound and nonbound cadmium differed in the two organs. The fraction not bound to metallothionein increased with time of exposure in both liver and kidney. However, total cadmium in the liver did not exceed potentially available binding sites of metallothionein, whereas total cadmium did exceed potentially available binding sites of metallothionein in the kidney where pathologic changes occurred. The results indicated that degeneration of cellular membranes is an early cellular effect of cadmium exposure followed later by toxicity to organelles, cellular necrosis, and interstitial fibrosis. Cadmium-induced cellular toxicity is more directly related to the fraction of cadmium in the kidney that is not bound to metallothionein than is total cadmium per se.

Evidence for effects of chronic lead exposure on blood pressure in experimental animals: an overview.

Information obtained in a number of experimental studies conducted over the last 40 years on the effects of lead on blood pressure is reviewed. Differences in animal species, age at beginning of exposure, level of lead exposure, indices of lead burden, and blood pressure effects of each study are reported. In several of the high-dose experiments, hypertension was observed, but nephrotoxicity of lead may have contributed to its development. Moreover, in other high-dose experiments, no hypertension was observed, and in at least one experiment, the evidence suggested that lead could reduce an elevated blood pressure. In contrast, the lower dose experiments consistently demonstrated a hypertensive effect. Overall, the data suggest a biphasic dose response. Establishment of an appropriate animal model to study blood pressure effects of lead will require careful assessment of dietary interactions with lead, unstressed blood pressure monitoring with standardized techniques, and documentation of biologically effective lead burden. Future research should examine lead exposure at more environmentally appropriate levels in order to determine the validity of associating this pollutant with blood pressure effects in the human population.

Effect of different levels and periods of lead exposure on tissue levels and excretion of lead, zinc, and calcium in the rat.

Influence of lead on tissue content and urinary excretion of lead, zinc, and calcium in rats was studied following various exposure periods. Weanling male rats were fed a trace mineral-sufficient diet with either 0, 200, 500, or 1000 ppm lead (as acetate) in drinking water for 4, 8, or 12 weeks. Blood lead ranged from 40 to over 100 micrograms/dl; kidney lead was highest at 4 weeks. Urinary lead excretion was highest at 4 weeks and declined with longer exposure. Urinary zinc excretion correlated positively with lead excretion at the lower excretion rates but plateaued at higher lead excretion rates. After 12 weeks exposure at each lead dose employed, decrease zinc concentration was observed in testes, bone, and brain. Plasma, erythrocyte, and kidney zinc were not affected, while pancreas and liver zinc were slightly elevated. Urine calcium was increased significantly only in rats exposed to 1000 ppm, possibly reflecting renal cell damage as determined by elevated renal calcium levels. These results indicate that lead dose is more important than exposure period for determining kidney lead levels, while urinary lead excretion rate is both dose and time dependent. Blood lead clearance values are relatively independent of dose and fall as exposure continues. Essential trace metal balance for zinc, especially, and to a lesser extent for calcium, is affected by the dose and length of chronic lead exposure.

Partitioning of renal zinc between metallothionein and ethylenediaminetetraacetic acid (EDTA) after treatment of rats with Ca(Na)2EDTA.

Calcium disodium edtate (EDTA) is a widely used chelator in the treatment of lead poisoning, although it also mobilizes other trace metals such as zinc. We have found that daily injection of 400 mg EDTA/kg body wt ip to rats results in an increase in the concentration of zinc (Zn) in the kidney when measured by atomic absorption spectroscopy. This increase is not associated with an induction of metallothionein (MT), a protein normally involved in the homeostasis of zinc. In fact, separation of kidney homogenate on a column of Sephadex G-75 demonstrates that virtually no Zn-MT remains in the kidney 24 hr after an EDTA injection, although after 3 days, Zn-MT levels appear to return to normal. Instead, increased renal Zn is found associated with residual EDTA. False high measurements of MT can be obtained from the Cd-saturation/hemoglobin assay due to the presence of residual EDTA in the kidney. Although nearly all of an injected dose of radiolabeled EDTA is rapidly excreted, 0.2 to 0.1% is retained in the kidney 1 day later; this is sufficient to account for the additional Zn. We propose that the small proportion of EDTA incorporated by pinocytosis into lysosomes in the renal cortical cells binds with all free Zn and subsequently depletes the MT concentration. Although the concentration of Zn measured in the kidney is elevated, there is actually less available for Zn-dependent activity.

Essential trace metal excretion from rats with lead exposure and during chelation therapy.

Urinary excretion of lead, zinc, calcium, magnesium, iron, copper, sodium, and potassium was measured in rats daily for 1 week after a 6-week exposure to 10,000 micrograms/ml lead in drinking water. Beginning on the third day, half of the lead-exposed and control rats were injected intraperitoneally with calcium disodium ethylenediaminetetraacetate (EDTA) daily for 3 days. Whole blood, plasma, and kidney metal concentrations were determined from samples obtained at the end of the experiment. Exposure to lead increased urinary excretion, not only of lead, but also of calcium, magnesium, zinc, copper, and iron. Excretion of sodium and potassium was not altered. Chelation therapy further increased excretion of lead, zinc, copper, and iron, but not magnesium. The increase in calcium excretion during chelation treatment (beyond that resulting from lead exposure per se) was accounted for by the Ca content of CaNa2-EDTA. EDTA treatment increased renal concentration of zinc but lowered renal concentration of lead, copper, and iron. These multimetal alterations may have implications for essential metal supplementation, particularly zinc, in persons being given chelation agents for excess lead exposure and in infants and children with low-level lead exposure not necessarily requiring chelation therapy.

Lead accumulation by rat renal brush border membrane vesicles.

Pb++ accumulation by rat renal cortical brush border membrane vesicles was evaluated by in vitro incubation with rapid filtration technique. Pb++ uptake was time- and concentration-dependent, with apparent saturation of binding sites at 100 to 200 microM (5 sec initial rate experiments). Equilibrium binding studies (60-min incubation) showed that the ratio of bound Pb++ to free Pb++ was constant at 1.25 +/- 0.07 between 0.01 to 10 microM Pb, with decreasing bound to free ratios at higher concentrations. Osmotic experiments showed that Pb++ uptake was due primarily to membrane binding rather than intravesicular accumulation. Electrochemical gradients of NaCl, KCl or protons did not increase vesicle uptake of Pb++. Incubation of vesicles with a number of amino acids did not stimulate Pb++ uptake although two (cysteine and glutathione) and the chelators EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) completely blocked this process. Competition studies with a number of other metals (at 10 microM and 1 mM) showed that only Sn++ or Sn +, La , Fe++ or Fe and Cu++ produced significant reductions in Pb++ uptake whereas Mg++, Ca++, Zn++, Cd++ and Hg++ were without effect on this process. Release of 203Pb from preloaded vesicles was accelerated in the presence of either cysteine or Sn +. Prior in vivo exposure to Pb (3 mg of Pb/kg i.v.) reduced Pb uptake to 70% of that of vesicles prepared from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin is a physiological inhibitor of urinary zinc excretion in anesthetized dogs.

Renal clearance experiments were performed on anesthetized dogs to determine the role of insulin in regulation of urinary zinc excretion. Intravenous infusion of somatostatin (2 micrograms/min) increased zinc excretion by approximately 100%, in association with 67% decreases in the plasma concentrations of both insulin and glucagon. Infusion of insulin (30 mU X kg-1 X min-1) along with the somatostatin maintained plasma insulin constant and completely eliminated the somatostatin-induced hyperzincuria; indeed, a small decrease in zinc excretion invariably occurred. Infusion of insulin alone (60 mU X kg-1 X min-1) decreased zinc excretion in five of six dogs. Plasma zinc concentration fell progressively, but to the same extent, throughout the experiment in all protocols. None of the hormonal infusions altered glomerular filtration rate, plasma concentrations of sodium, calcium, or magnesium or urinary excretion of these cations. We conclude that insulin, at physiological plasma concentrations, exerts an inhibitory effect on urinary zinc excretion.

Plasma renin is increased in young rats exposed to lead in utero and during nursing.

Rats were exposed continuously to Pb in utero and after birth by giving their mothers, during pregnancy and lactation, drinking water containing 0, 5, 25, 100, or 500 ppm Pb (as Pb acetate); they were sacrificed at 1 month of age, at which time their mean blood Pb concentrations were, respectively, approximately 3, 9, 19, 30, and 70 micrograms/dl. All Pb-exposed groups sacrificed by decapitation had elevated mean plasma renin activities (PRA), relative to controls. Pentobarbarbital-anesthesia and laparotomy markedly increased PRA in the 0, 100, and 500 ppm groups, but the increase was significantly less in the 100 ppm group. Renal renin concentration was normal in the 5 and 25 ppm groups, but was significantly increased in the 100 and 500 ppm groups. The ratio of plasma angiotensin II to PRA was normal in the 100 ppm group but significantly reduced in the 500 ppm group. We conclude that exposure of rats in utero and during lactation to doses of Pb which produce blood Pb concentrations similar to those generally present in human populations stimulates basal renin secretion in 1-month-old rats, but partially inhibits the response to renin-releasing stimuli. The highest dose reduces plasma angiotensin II at any given PRA. These results, taken with previous publications, emphasize that the effects of lead on plasma renin even within a single species are greatly affected by the timing of the exposure.

Lead, hypertension, and the renin-angiotensin system in rats.

Rats were exposed continuously to Pb in utero and after birth by giving their mothers, during pregnancy and lactation, drinking water containing 0, 100, or 500 ppm Pb (as Pb acetate) and then continuing this regimen after weaning. Male rats received 100 ppm developed a significant elevation of systolic blood pressure (152 +/- 3.7 mm Hg vs. 135 +/- 5.6 for controls) at 3 1/2 months and remained hypertensive until sacrifice at 6 months; 500 ppm rats remained normotensive. Both 100 ppm and 500 ppm females remained normotensive. At 6 months, PRA was significantly reduced in the 100 ppm male group but was normal in the 500 ppm group. There were dose-dependent decreases in the AII/PRA ration and in renal renin. Pulmonary converting enzyme activity was not changed by Pb exposure. Blood [Pb] was 40 and 71 mug/dl, respectively, and kidney [Pb] was 4.8 and 22.9 mug/gm. Renal histology was normal in the 100 ppm group. We conclude that doses of Pb which produce blood [Pb] seen in many people are capable of inducing modest hypertension in male rats; higher doses fail to do so. The hypertension is associated with a reduction in PRA and All and therefore is unlikely to be due to hyperactivity of the RAS. (J Lab Clin Med 99:354, 1982.)

Urinary excretion of zinc and iron following injection of bacteria in the unanesthetized rabbit.

The effects of injection of bacteria (Pasteurella multocida) on the urinary excretion of zinc and iron (during the period when plasma concentrations of these trace metals were falling) were determined in unanesthetized New Zealand white rabbits. During the initial 2 h following injection with bacteria there was a trend towards an increased urinary excretion of zinc and iron. This short-term increase in zinc and iron excretion, however, could only account for a small percentage of the reduction in plasma concentrations of these trace metals that occurs during infection. We conclude that most of the reduction in plasma iron and zinc that occurs during infection is attributable to a redistribution within the body and not to increased excretion.

Lead increases urinary zinc excretion in rats.

The major purpose of this study was to determine whether acute or chronic Pb exposure would increase urinary excretion of zinc in the rat. Four groups of unanesthetized rats were given 0, 0.03, 0.3, or 3 mg Pb (as acetate) kg intravenously, and urinary excretion of zinc, sodium, and potassium was monitored for 6 h. Only at the highest dose was urinary Zn excretion significantly elevated; there were no significant changes in sodium and potassium excretion at any dose. Two other groups of rats were studied for 9 weeks in metabolism cages before and during administration of either 500 ppm Pb (as acetate) or equimolar Na acetate in the drinking water. Two days after Pb treatment and continuing through day 35, Zn excretion was elevated in the Pb-exposed animals; beyond this day, zinc excretion became similar in the two groups. The difference in Zn excretion was not the result of lower water intake by the Pb-treated animals. At sacrifice (70 days after starting Pb exposure), Pb-exposed animals had lower Zn content of the plasma and testis, but there was no difference in kidney Zn. Plasma renin activity was significantly higher in Pb-exposed animals. We conclude that chronic Pb exposure in rats can result in some degree of decreased tissue zinc, which is, at least in part, secondary to increased urinary losses of zinc.

Renal tubular handling of zinc in the dog.

Clearance and stop-flow techniques, performed on anesthetized dogs, were used to obtain evidence for renal tubular reabsorption or secretion of 65Zn (administered intravenously with varying amounts of carrier ZnCl2). Ultrafilterability of plasma 65Zn was determined in vitro with Amicon CF-50A filter cones. Clearances were obtained under six conditions: antidiuresis, mannitol diuresis alone and following the infusion of chlorothiazide, citrate, cysteine, histidine. The percentage of ultrafilterability averaged, respectively, 1.15, 1.38, 0.91, 1.53, 19.90, and 11.61, while fractional zinc excretion averaged 0.046, 0.10, 0.31, 0.43, 0.78, and 0.12, respectively. Control stop-flow patterns invariably showed net Zn reabsorption in the distal nephron. Stop-flow patterns after chlorothiazide revealed significant net secretion in the far distal area in all animals. Patterns after either citrate or histidine infusion revealed significant net reabsorption in the proximal nephron. Those after cysteine were similar to the control patterns. Thus, both clearance and stop-flow experiments revealed net reabsorption as the major tubular mechanism of zinc handling, tubular secretion occurring only during chlorothiazide administration.

In vivo and in vitro effects of lead on vascular reactivity in rats.

The effects of lead on vascular responsiveness were examined in rats. Adult rats, which had received levels of lead acetate in their drinking water to produce blood levels similar to those seen in some urban human populations, consistently had higher systolic blood pressures compared to age-matched controls. Helical strips of tail arteries from the lead-treated rats displayed a greater force-generating ability in response to the cumulative addition of methoxamine to the muscle bath. There were no differences in ED50 between the two groups. Similar results were obtained when norepinephrine was used. The calcium-entry blocker, D 600, was less effective in reducing in reducing contractions induced by methoxamine in lead-treated rats than in controls. There were no differences between the two groups in responses to KCl or electrical stimulation of nerve endings. Contractile responses to norepinephrine, methoxamine, KCl, and nerve stimulation in arteries from untreated rats were unaltered by addition of lead acetate to the muscle bath. These results demonstrate that hypertension induced by moderate levels of lead intake is associated with an increased vascular responsiveness to alpha-adrenergic agonists.

A receptive period for estradiol-induced luteolysis in the rhesus monkey.

This study was performed to test the hypothesis that responsiveness to the luteolytic action of estradiol is acquired as the luteal phase of the menstrual cycle progresses. The luteolytic effect of fixed estradiol increment (270 +/- 12 pg/ml serum) was assessed at different stages of luteal function in rhesus monkeys. A 4-day elevation in estradiol early in the luteal phase (days 2--6 after the LH peak) caused a decrease in the concentration of serum progesterone but did not shorten luteal life span. In contrast, when provided during the midluteal phase (days 6--10), the same 4-day estradiol increment promptly induced premature luteolysis. Furthermore, during sustained exposure to the extradiol increment from days 2--10, signs of premature luteolysis were not evident until day 7 after the LH peak. Thus, the effects of estradiol early in the luteal phase do not alter luteal life span; it is the effects after day 6 that precipitate luteolysis. These observations support the existence of a receptive period for the luteolytic action of estradiol in the rhesus monkey.

Effect of glucagon on zinc excretion in anesthetized dogs.

The present studies were performed on anesthetized dogs to determine whether urinary zinc excretion is altered when plasma glucagon concentration is elevated acutely. Glucagon infusion (5 ng.kg-1.min-1) adequate to double base-line plasma glucagon significantly increased zinc excretion in a reversible manner; plasma zinc concentration was unchanged. A larger dose of glucagon (50 ng.kg-1.min-1) produced no significantly greater effect. Clearance experiments using 65Zn to measure ultrafilterable zinc concentration revealed no change in ultrafilterable zinc during glucagon infusion. In a third series of experiments, acetylcholine (25 micrograms/min) was infused into one renal artery while zinc excretion and renal function of the two kidneys were compared. Acetylcholine did not significantly alter zinc excretion despite large changes in sodium and water excretion. The changes in zinc excretion observed in all experiments correlated well with glomerular filtration rate changes, but additional contributions of changes in tubular handling of zinc cannot be ruled out.