Effects of nickel chloride on reproduction of the rat and possible antagonistic role of selenium.

Nickel (10-100 ppm added as NiCl2) was studied to determine its effects on reproduction of Wistar rats. In nine experimental groups, females, males or both were exposed to nickel in drinking water. In one female group and one male group, the drinking water was also supplemented with 0.3 ppm selenium (added as Na2SeO3). Breeding success and the growth and viability of pups were recorded. Nickel, copper and zinc concentrations in kidneys, liver and skin (with fur) of the females, males and pups were determined with an atomic absorption spectrophotometer. In addition, histology of the male testes (from control and nickel-exposed groups) was studied. The female exposures started 14, 28 or 100 days before copulation and continued during pregnancy and lactation. When the males were exposed (for 28 or 42 days before copulation), NiCl2 reduced both the number of pregnancies and the number of pups born. In the testes, NiCl2 induced shrinkage of the seminiferous tubules, which seemed to close some of the tubules. In the tubules, NiCl2 decreased the number of basal spermatogonia. When the females or both parents were exposed to NiCl2, pup mortality during lactation was high. However, when the females were drinking NiCl2 supplemented with selenium, all the pups survived and development of the total mass of the litters was even better than in the control group. In the same way, in males, selenium supplementation of the drinking water protected those pups that were born; but fertility was lower than with the control treatment. In the tissues studied, nickel accumulated most in the kidneys and then in the liver and skin. In each type of organ, there was a clear dose response relationship. In the pups, in particular, selenium (given to the females) increased the amount of nickel in tissues compared with corresponding administration of nickel without selenium. In summary, selenium seemed to counteract the deleterious effects of NiCl2 on the reproduction of rats.     

Dose-dependent mercuric chloride tubular injury in rat kidney

Mercuric chloride (HgCl 2 ) produces an acute renal failure in experimental animal models. Since the mechanism of tubular injury has not completely been clarified, this morpho-quantitative study was undertaken to better understand the effects of 2 sublethal doses (T1=1 mg/kg and T3.5=3.5 mg/kg) of HgCl 2 in rat proximal tubules. Morphometrical analysis was performed to quantify both cytoplasmic and nuclear changes found in treated in respect to saline-injected proximal tubules. In the controls, single-cell damage was occasional and nucleolar changes were absent. HgCl 2 induced progressively severe proximal tubule atrophy. In the T1 group, necrosis was limited to pars recta cells and nucleolar segregation was often partial. In the T3.5 group, atrophy was extensive in both convoluted and straight tracts, the nucleolus was completely segregated and coiled body-like inclusions were detached from it. Ultrastructural analysis confirmed dose-dependent changes within straight proximal tubules, i.e., necrosis, apoptosis, nucleolar segregation, swollen mitochondria, vacuolization, and disrupted brush border. In conclusion, in the rat kidney HgCl 2 induced dose-dependent alterations not only in the cytoplasm but also in the nucleus of proximal tubule cells. These findings will be useful for better understanding of the pathogenesis of mercury nephrotoxicity and its genotoxic effect.     

Histochemical and immunocytochemical evaluation of copper and metallothionein in the liver and kidney of copper-loaded rats

Histochemical methods do not always show a good correlation with analytical measurement of copper content and consequently immunoreactive staining techniques for metallothionein (MT) have recently been employed for the differential diagnosis of copper-associated diseases. This study compares histochemical with immunocytochemical methods for the assessment of copper status. Male rats were fed a high copper (1 g/kg) diet for 16 weeks and killed sequentially during this period. The livers and kidneys were analysed for copper and zinc (atomic absorption spectrophotometry), and sections were stained with rubeanic acid and rhodanine for copper and for immunoreactive MT using the DNP localization system. Immunoreactive stains for MT corresponded better with copper content than histochemical stains and were more sensitive, albeit less selective, indicators of copper accumulation. Moreover, major differences in intracellular staining were apparent between the two methods, attributed to differences in copper binding and microcompartmentalization of metal.     

Use of sodium diethyldithiocarbamate in the treatment of nickel carbonyl poisoning

This presentation is primarily concerned with the use of sodium diethyldithiocarbamate (Dithiocarb) in the treatment of acute nickel carbonyl poisoning. Reference will be made to earlier studies pertaining to diagnosis in relation to treatment. In addition, recent studies will be briefly considered on the administration of Dithiocarb to rats developing tumors following the muscular implantations of nickel subsulfide (Ni3S2).     

Isolated perfused rat kidney and liver combined

In the late phase of the fever occurring 120 or more min after i.v. injection of endotoxin (1 ug/kg) to female rabbits, marked shifts of thresholds for respiratory evaporative heat loss and for peripheral vasodilatation to higher body core temperatures were observed. In contrast, the threshold body core temperature for cold thermogenesis was shifted downwards. As a result, the interthreshold zone was widened. Within the body temperature range of 37.4 to 39.9°C neither heat production or heat loss mechanisms were operant and the body temperature was determined mainly by passive heat transfer between the body and the environment. Outside this zone, the sensitivities of the heat and cold deference activities to changes in body core temperature appeared to be unchanged.     

Effect of reduced chloride reabsorption on renin release in the isolated rat kidney

To investigate the relationship between tubular reabsorption of chloride and renal renin release in the isolated perfused rat kidney, perfusate renin activity was measured during substitution of either nitrate or thiocyanate for varying amounts of perfusate chloride but with maintained perfusate sodium concentration. Renin rose significantly as perfusate chloride fell; there was a sevenfold increase between perfusion with normal chloride and almost complete substitution of chloride by nitrate. With a normal perfusate chloride the addition of furosemide 10^–4 M to the perfusate also led to an increase in renin and a reduction in tubule chloride reabsorption. For all these experiments there was a significant negative correlation between renin and absolute tubular reabsorption of chloride (r=–0.68,P<0.001), but no such relationship with absolute sodium reabsorption. Renin release in a nonfiltering kidney, produced by elevating perfusate albumin concentration, increased approximately 40-fold. Thus increasing plasma oncotic pressure elevates renin by mechanisms additional to cessation of tubular chloride absorption. However, substitution of chloride in the perfusate by nitrate in this nonfiltering kidney did not further elevate renin release. We conclude that renin release is influenced by a signal dependent on, and inversely proportional to, chloride reabsorption in the thick ascending limb of the Loop of Henle.     

Estrogen modulates ClC-2 chloride channel gene expression in rat kidney

ClC-2 is a CLC family member of chloride channels sensitive to changes in cell volume, pH and voltage. The ClC-2 is widely distributed along the nephron although in the kidney its role still not well understood. Aldosterone studies suggest that ClC-2 expression in the kidney may be hormonally regulated. To explore the possibility that estrogen control ClC-2 expression, we investigated whether its expression changed in the kidney of female Wistar rats subjected to ovariectomy with or without near-physiological or high doses of 17-estradiol benzoate treatment for 10 days. Total RNA isolated from rat kidney and dissected nephron segments was analyzed by ribonuclease protection assay and/or a semi-quantitative RT-PCR. The renal ClC-2 protein expression was analyzed by Western blot. The decreased renal expression of ClC-2 mRNA and protein observed in ovariectomized rats was restored to control levels after treatment with low doses of estradiol. Higher dose estradiol lead to an even greater increase in ClC-2 mRNA and protein expression. This change in overall expression was shown to be caused by the modulation of ClC-2 mRNA expression in the proximal tubule. These results suggest that ClC-2 may be involved in estrogen-induced Cl^– transport in rat kidney.     

The intracellular chloride activity of rat kidney proximal tubular cells

The intracellular Cl^? activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl^? sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measureV _Cl and using — in separate experiments — conventional KCl-filled microelectrodes to measure the membrane potential,V _m. After correction for interference from other anions onV _Cl the intracellular Cl^? activity averaged 13.1 mmol·l^?1 SD±4.5 mmol·l^?1 (n=96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl^? activity of 90–103 mmol·l^?1 andV _m of ?71.2 mV, SD±4.9 mV (n=23) to amount to 6.3 to 6.7 mmol·l^?1. Since both cell membranes are permeable for Cl^? ions, as concluded from luminal and/or peritubular Cl^? substitution experiments, we conclude that the cellular Cl^? accumulation above equilibrium results from transcellular active Cl^? transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl^? concentration after substitution of luminal Cl^? by gluconate, however, we deduce that transcellular Cl^? absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl^? flux is passive and paracellular.     

Effects of chronic oral nickel chloride administration on glycaemia and renal function in normal and diabetic rats

Effects of nickel on glycaemia are conflicting. We have investigated the effects of oral administration of nickel chloride for 5 weeks on glycaemia and renal function in normal and streptozotocin-diabetic rats. The results show increase (P< 0.05) in plasma glucose and sodium and decrease (P< 0.05) in insulin concentrations only in normal experiment by comparison with normal control. Plasma potassium concentration was elevated (P< 0.05) only in diabetic experiment by comparison with diabetic control. Plasma urea levels were raised (P< 0.05) in both groups of experiments by comparison with respective controls and plasma creatinine level was elevated (P< 0.05) only in diabetic experiment. GFR was reduced (P< 0.05) only in normal experiment by comparison with control. Kidney weights in normal and diabetics were not effected by nickel chloride administration. Total food and water intakes in normal and diabetic experiments were lower by comparison with respective controls. These were accompanied by failure to increase body weights. In addition, total urine volume and sodium were reduced in normal and diabetic experiments. Urine potassium was lower only in normal experiment by comparison with normal control. We conclude that chronic nickel chloride administration induces hyperglycemia possibly through reduction in blood insulin levels and could be toxic to renal function.     

Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and its changes during development

The distribution of metallothionein (MT) during human development was investigated using both immunohistochemical and biochemical methods. The level of MT in the fetal liver was higher than the adult liver levels. Higher levels of zinc (Zn) and copper (Cu) were also detected in the fetal liver compared to the adult liver. Although cadmium (Cd) was present in detectable levels in the human adult liver, none was detected in the human fetal liver. MT was localized in the nucleus and the cytoplasm of human fetal and neonatal hepatocytes, using a specific rabbit antibody raised to rat liver MT. In the adult human liver cells, MT was localized mainly in the cytoplasm. In the fetal and neonatal human kidney, MT was localized mainly in the nucleus and the cytoplasm of the proximal tubular epithelial cells. In the adult kidney, in addition to nuclear-cytoplasmic localization of MT, intraluminal localization was also observed.     

Effects of chloride transport inhibition and chloride substitution on neuron function and on hypoxic spreading-depression-like depolarization in rat hippocampal slices

Chloride fluxes play a crucial role in synaptic inhibition, cell pH regulation, as well as in cell volume control. In many neuropathological processes, cell swelling is a pivotal parameter, since cell volume changes and the dimension of the interstitial space critically modulate synchronized neuronal activity as well as the tissue's susceptibility to seizures or spreading depression. This study therefore focuses on the effects of different Cl(-) transport inhibitors and Cl(-) substitution on neuronal function and hypoxia-induced changes in rat hippocampal tissue slices. Orthodromically evoked focal excitatory postsynaptic potentials were depressed by furosemide (2mM), 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (1mM) and Cl(-) substitution by methylsulfate, but were enhanced by 4,4'-dinitrostilbene-2,2'-disulfonic acid (1mM). All four treatments induced multiple population spike firing in response to single orthodromic volleys, suggesting reduced synaptic inhibition. Antidromic population spikes increased following Cl(-) withdrawal, were unaffected in the presence of furosemide and 4, 4'-dinitrostilbene-2,2'-disulfonic acid, but were abolished by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The amplitude of the hypoxic spreading-depression-like extracellular potential shift was reduced by furosemide, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and Cl(-) withdrawal, i.e. by the same treatments that depressed orthodromically evoked postsynaptic potentials. Furosemide prolonged the time to onset and the duration of the spreading-depression-like extracellular potential shift, while 4, 4'-dinitrostilbene-2,2'-disulfonic acid shortened the time to onset. Spreading-depression-related cell swelling was recorded as the shrinkage of relative interstitial space, which was measured as tetramethylammonium-chloride space. Neither the Cl(-) transport inhibitors nor Cl(-) withdrawal had any detectable effect on spreading-depression-related cell swelling. CA1 pyramidal neurons usually hyperpolarized during drug application and their input resistance decreased. Cl(-) withdrawal increased their input resistance and caused spontaneous burst firing. Hypoxia caused the expected spreading-depression-like rapid, near complete depolarization of single pyramidal neurons and drastically reduced their input resistance. The three Cl(-) transport inhibitors and Cl(-) withdrawal delayed the onset of the hypoxic depolarization. In low Cl(-) solutions, the apparent threshold potential at which spreading depression was triggered shifted to more positive membrane potentials. The final voltage of the hypoxic depolarization was, however, not affected.It appears from these results that the reduction in the hypoxic spreading-depression-like extracellular potential shifts by Cl(-) transport inhibitors is at least partially attributable to desynchronization of depolarization, not to decreased depolarization in individual cells. Other contributing factors could be changes in recording conditions, depression of swelling-induced amino acid release from glial cells and unspecific side-effects of the applied drugs. Desynchronization could also account for the delayed spreading-depression onset. It is concluded that Cl(-) fluxes play a role in the triggering of spreading depression, but the spreading-depression-like depolarization itself or its self-regenerative character is not mediated by Cl(-).     

Hepatic toxicity of nickel chloride in rats

Enhanced lipid peroxidation was observed in livers of rats killed 24 hr after sc injection of nickel chloride (NiCl2) (750 mumol per kg), as evidenced by 13-fold increase of conjugated dienes in microsomal lipids and 4-fold increase of thiobarbituric acid (TBA) chromogens in hepatic cytosol. Histologic examination of livers from rats killed one to three days after NiCl2 injection (500 mumol per kg) showed microvesicular fatty metamorphosis, mild hydropic degeneration, and foci of inflammation. Microvesicular steatosis of hepatocytes was confirmed by electron microscopy. Dose-related increases of serum aspartate aminotransferase (ALT) activity (up to 7-fold vs controls) and alanine aminotransferase (ALT) activity (up to 3-fold vs controls) were observed 24 hr after injection of NiCl2 (125 to 750 mumol per kg); diminished serum alkaline phosphatase activity (up to 72 percent reduction vs controls) was seen at NiCl2 dosages from 375 to 750 mumol per kg. Diethyldithiocarbamate did not influence the effects of NiCl2 on TBA-chromogens in liver homogenates or on serum AST and ALT activities but acted synergistically with NiCl2 to diminish serum alkaline phosphatase activity and to increase serum bilirubin concentration. This study demonstrates that parenteral administration of NiCl2 to rats produces acute hepatic toxicity, with enhanced lipid peroxidation, microvesicular steatosis, and increased serum AST and ALT activities.     

Enzyme changes in the brain, liver and kidney following repeated administration of mercuric chloride

Alterations in the levels of selected enzymes have been studied in the liver, kidney and brain of mouse following mercuric chloride (1 mg/Kg body wt./d) administration for 10, 20 and 30 d. The activity of acid phosphatase increased in all the tissues, the highest increase was recorded in the kidneys which showed as much as 4.5 fold elevation following mercuric chloride administration for 30 d. Although the alkaline phosphatase activity in the liver and the brain increased following HgCl2 administration, the kidneys experienced a tremendous decline in this enzyme following the same treatment. Mercury-induced changes in ATPase were complex inasmuch as the nature and magnitude of these changes varied with the tissue as well as the duration of the treatment. Whereas the liver ATPase declined after all the treatment intervals, this enzyme increased in the kidney and brain following administration of HgCl2 for 10 d. However, both the kidneys and brain registered a substantial fall in ATPase activity when HgCl2 administration was continued for 30 d. The levels of both glucose-6-phosphatase and succinic dehydrogenase decreased in all the tissues following HgCl2 administration. Invariably, the magnitude of decrease was the highest after 30 d treatment with HgCl2.     

Electrochemical forces for chloride transport in the proximal tubules of the rat kidney

The electrochemical forces for chloride transport in the proximal tubule of the rat kidney were studied using micropuncture techniques. Electrical transmembrane potentials were recorded in randomly punctured tubules with Ling-Gerhard electrodes. Chloride activities in the luminal, cellular and interstitial compartments were measured with ion selective micro-electrodes. Electrical potential measurements between cell to interstitium and lumen to interstitium were -72.1 ± 2.6 mVand ± 0.5±1.4mV(mean ± S.D.)respectively. The calculated chloride concentrations for lumen, cell and interstitium were 133.0±10.3 mM, 8.5±1.0 mM and 99.1 ± 3.2 mM (mean ± S.D.) respectively. The net electrochemical forces, qualitatively, offer a passive chloride ion pathway through the tubular wall and a chloride equilibrium over the luminal membrane seems to exist.     

Differential gene expression in nickel(II)-treated normal rat kidney cells

Nickel(II) compounds are carcinogenic metals which induce genotoxicity and oxidative stress through the generation of reactive oxygen species. In search of new molecular pathways toward understanding the molecular mechanism of nickel(II)-induced carcinogensis, we performed mRNA differential display analysis using total RNA extracted from nickel(II) acetate-treated normal rat kidney cells (NRK-52E). Cells were exposed for 2 months to 160 and 240 microM nickel(II) concentrations. cDNAs corresponding to mRNAs for which expression levels were altered by nickel(II) were isolated, sequenced, and followed by a GenBank Blast homology search. Specificity of differential expression of cDNAs was determined by RT-PCR. Two of them (SH3BGRL3 and FHIT) were down-regulated and one (metallothionein) was up-regulated by nickel(II) treatment. The expression of these mRNAs were nickel(II) concentration-dependent. Overall, although the fundamental questions related to function of these genes in nickel(II)-mediated carcinogenicity are not answered, our study suggests that they can be interesting candidates for studies of molecular mechanisms of nickel(II) carcinogenesis.     

Effects of ammonium chloride on synaptic transmission in the rat hippocampal slice

Effects of ammonia on excitatory synaptic transmission were studied in the rat hippocampal slice preparation. Population spikes, elicited by orthodromic or antidromic stimulation, were recorded in the cell body layer of the CA1, CA3 and dentate regions. Perfusion with 5 mM ammonium chloride induced a profound and reversible depression of orthodromically evoked population spikes in all three regions. Antidromic population spikes were not depressed in any of the regions, indicating that neither axonal conduction nor electrical excitability were affected by ammonia. The paired-pulse test revealed a transient disinhibition during the early phase of perfusion. Iontophoretic application of glutamate evoked unit firing even when the synaptically evoked responses were reduced by ammonia, indicating that the postsynaptic sensitivity to the putative transmitter was not depressed. Depression of release of the excitatory transmitter, probably because of depletion following the block of transmitter synthesis, is the likely explanation of these findings. It is suggested that ammonia-induced depression of excitatory transmission may account for coma and other symptoms of central nervous system depression encountered in hyperammonemic states.     

Aldosterone and high-NaCl diet modulate ClC-2 chloride channel gene expression in rat kidney

It is well known that Na+ reabsorption in the kidney can be regulated by aldosterone. Although Cl- is the most abundant anion present in the extra cellular fluids the involvement of aldosterone in the regulation of Cl- conductance through Cl- channels at the molecular level is unknown. In this study, the effects of aldosterone and high-Na+ diet on the expression of ClC-2, a cell volume-, pH- and voltage-sensitive Cl- channel, was examined in the rat kidney. Total RNA isolated from Wistar rats fed a high-Na+ diet for 5 days, furosemide treatment, adrenalectomy and adrenalectomy with replacement of normal plasma levels of aldosterone were compared by the use of ribonuclease protection assay (RPA), and/or a semi-quantitative RT-PCR. The high-Na+ diet reduced renal mRNA and protein ClC-2 expression. The renal expression of ClC-2 mRNA decreased in adrenalectomized rats and was restored by plasma aldosterone replacement. In addition, the semi-quantitative RT-PCR in different segments of the nephron showed that these changes were secondary to the modulation of ClC-2 mRNA expression by aldosterone in the cortical and medullary segments of thick ascending limbs of Henle's loop. These results suggest that ClC-2 may be involved with aldosterone-induced Cl- transport in the kidney.