Role of PI3K-Akt and MAPK Signaling in Uranyl Nitrate-Induced Nephrotoxicity.

Uranium is a heavy metal of considerable environmental and occupational concern. It is well-known that the kidney is the major target organ of uranium exposure. Elucidating the mechanistic basis of uranium interactions is essential for monitoring the health risk. In the present study, we investigated the cellular mechanisms involved in uranyl nitrate-induced nephrotoxicity. Male Swiss albino mice were administrated with a single intraperitoneal dose of 2 and 4 mg/kg of uranyl nitrate at different time points 1, 3, 5, 7, 14, and 28 days. Uranyl nitrate intoxication-induced apoptosis in the kidney tissue was observed by TUNEL assay. To assess the proliferation, immunohistochemistry was performed using Ki67 proliferative marker followed by western blotting to confirm the involvement of key signaling molecules. The number of TUNEL positive nuclei peaked at third day after uranyl nitrate insult. The increased expression of proliferation marker Ki67 suggests the enhanced DNA repair process prominently at seventh day. Uranyl nitrate administration also resulted in activation of extracellular signal-regulated kinases (ERK), Akt, and c-Jun N-terminal kinases (JNK) expression. All these changes were found to be time-dependent. The result of the current study suggests that uranyl nitrate induces acute renal injury by activation of apoptosis through JNK pathway, while the early activation of signaling molecules Akt and ERK promotes the tubular cell proliferation and cell survival.

Radiation exposure from depleted uranium: The radiation bystander effect.

Depleted uranium (DU) is a radioactive heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. In vivo studies have also demonstrated that DU is leukemogenic and genotoxic. DU possesses both a radiological (alpha particle) and chemical (metal) component but is generally considered a chemical biohazard. Studies have shown that alpha particle radiation does play a role in DU's toxic effects. Evidence has accumulated that non-irradiated cells in the vicinity of irradiated cells can have a response to ionization events. The purpose of this study was to determine if these "bystander effects" play a role in DU's toxic and neoplastic effects using HOS cells. We investigated the bystander responses between DU-exposed cells and non-exposed cells by co-culturing the two equal populations. Decreased cell survival and increased neoplastic transformation were observed in the non-DU exposed cells following 4 or 24h co-culture. In contrast Ni (II)- or Cr(VI)- exposed cells were unable to alter those biological effects in non-Ni(II) or non-Cr(VI) exposed co-cultured cells. Transfer experiments using medium from the DU-exposed and non-exposed co-cultured cells was able to cause adverse biological responses in cells; these results demonstrated that a factor (s) is secreted into the co-culture medium which is involved in this DU-associated bystander effect. This novel effect of DU exposure could have implications for radiation risk and for health risk assessment associated with DU exposure.

Molecular, cellular, and tissue impact of depleted uranium on xenobiotic-metabolizing enzymes.

Enzymes that metabolize xenobiotics (XME) are well recognized in experimental models as representative indicators of organ detoxification functions and of exposure to toxicants. As several in vivo studies have shown, uranium can alter XME in the rat liver or kidneys after either acute or chronic exposure. To determine how length or level of exposure affects these changes in XME, we continued our investigation of chronic rat exposure to depleted uranium (DU, uranyl nitrate). The first study examined the effect of duration (1-18 months) of chronic exposure to DU, the second evaluated dose dependence, from a level close to that found in the environment near mining sites (0.2 mg/L) to a supra-environmental dose (120 mg/L, 10 times the highest level naturally found in the environment), and the third was an in vitro assessment of whether DU exposure directly affects XME and, in particular, CYP3A. The experimental in vivo models used here demonstrated that CYP3A is the enzyme modified to the greatest extent: high gene expression changed after 6 and 9 months. The most substantial effects were observed in the liver of rats after 9 months of exposure to 120 mg/L of DU: CYP3A gene and protein expression and enzyme activity all decreased by more than 40 %. Nonetheless, no direct effect of DU by itself was observed after in vitro exposure of rat microsomal preparations, HepG2 cells, or human primary hepatocytes. Overall, these results probably indicate the occurrence of regulatory or adaptive mechanisms that could explain the indirect effect observed in vivo after chronic exposure.

Reduced clearance of ε-acetamidocaproic acid in rats with acute renal failure induced by uranyl nitrate.

OBJECTIVES: Anti-ulcer drugs are frequently used in patients with acute renal failure (ARF). Zinc acexamate is ionized to zinc and ε-acetamidocaproic acid and free EACA exerts a potent therapeutic effect in treating gastric or duodenal ulcers with few side effects. Thus, pharmacokinetic changes in rats with acute renal failure induced by uranyl nitrate (U-ARF rats) were investigated in this study. METHODS: The in-vivo pharmacokinetics and in-vitro hepatic/intestinal metabolism of EACA were assessed using control and U-ARF rats. The mechanism of urinary excretion of EACA was further investigated in rats. KEY FINDINGS: After intravenous and oral administration of zinc acexamate to U-ARF rats, there were significant increases in the values of the area under the curve (AUC) and decreases in the values for time-averaged renal and nonrenal clearances (Cl(r) and Cl(nr) , respectively) compared with control rats. Slower Cl(nr) was partly due to a decrease in the metabolism in liver and/or intestine. Slower Cl(r) could have been due to urine flow rate-dependent timed-interval renal clearance, decrease in organic anion transporter-mediated renal excretion (drug interaction with probenecid and decrease in the relative contribution of net secretion compared with glomerular filtration in U-ARF rats) and/or impaired kidney function. CONCLUSIONS: The pharmacokinetics were significantly altered in U-ARF rats due to the changes in both the hepatic/intestinal metabolism and urinary excretion.

Uranyl nitrate-exposed rat alveolar macrophages cell death: influence of superoxide anion and TNF α mediators.

Uranium compounds are widely used in the nuclear fuel cycle, military and many other diverse industrial processes. Health risks associated with uranium exposure include nephrotoxicity, cancer, respiratory, and immune disorders. Macrophages present in body tissues are the main cell type involved in the internalization of uranium particles. To better understand the pathological effects associated with depleted uranium (DU) inhalation, we examined the metabolic activity, phagocytosis, genotoxicity and inflammation on DU-exposed rat alveolar macrophages (12.5-200 µM). Stability and dissolution of DU could differ depending on the dissolvent and in turn alter its biological action. We dissolved DU in sodium bicarbonate (NaHCO3 100 mM) and in what we consider a more physiological vehicle resembling human internal media: sodium chloride (NaCl 0.9%). We demonstrate that uranyl nitrate in NaCl solubilizes, enters the cell, and elicits its cytotoxic effect similarly to when it is diluted in NaHCO3. We show that irrespective of the dissolvent employed, uranyl nitrate impairs cell metabolism, and at low doses induces both phagocytosis and generation of superoxide anion (O2⁻). At high doses it provokes the secretion of TNFα and through all the range of doses tested, apoptosis. We herein suggest that at DU low doses O2⁻ may act as the principal mediator of DNA damage while at higher doses the signaling pathway mediated by O2⁻ may be blocked, prevailing damage to DNA by the TNFα route. The study of macrophage functions after uranyl nitrate treatment could provide insights into the pathophysiology of uranium-related diseases.

Toxicity of depleted uranium complexes is independent of p53 activity.

The p53 tumor suppressor protein is one of the key checkpoints in cellular response to a variety of stress mechanisms, including exposure to various toxic metal complexes. Previous studies have demonstrated that arsenic and chromium complexes are able to activate p53, but there is a dearth of data investigating whether uranium complexes exhibit similar effects. The use of depleted uranium (DU) has increased in recent years, raising concern about DU's potential carcinogenic effects. Previous studies have shown that uranyl acetate and uranyl nitrate are capable of inducing DNA strand breaks and potentially of inducing oxidative stress through free radical generation, two potential mechanisms for activation of p53. Based on these studies, we hypothesized that either uranyl acetate or uranyl nitrate could act as an activator of p53. We tested this hypothesis using a combination of cytotoxicity assays, p53 activity assays, western blotting and flow cytometry. All of our results demonstrate that there is not a p53-mediated response to either uranyl acetate or uranyl nitrate, demonstrating that any cellular response to uranium exposure likely occurs in a p53-independent fashion under the conditions studied.

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney.

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.

A case of acute radiation syndrome from the dermatological aspect.

Accidental whole-body overexposure of radiation occurs very rarely. Radiation exposure causes DNA breaks in the cells and shows various clinical features, which are time dependent, dose dependent and tissue dependent. Neutron rays are more destructive than gamma rays but their actual effect on humans have been under-reported. We observed the time-dependent and the dose-dependent dermatological changes in a patient who was severely irradiated by neutron and gamma rays, with the aim of clarifying the clinicopathological features of severely irradiated skin. The detection of DNA breaks in keratinocytes was performed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique. The degenerative changes of the skin and the re-epithelialization varied in a time dependent and dose dependent manner. DNA breaks were significantly higher in irradiated keratinocytes. Neutron rays caused depth-dependent degeneration of the skin. Evaluation of DNA breaks in the skin cells might be a clue to estimate local dosimetry.

Acute exposure of uranyl nitrate causes lipid peroxidation and histopathological damage in brain and bone of Wistar rat.

Although the kidneys are the main target organs for uranium (U) toxicity, recent studies have shown that U can cross the blood-brain barrier to accumulate in the brain. Uranyl nitrate (U-238)induced oxidative damage was investigated in brain and bone of Wistar rats after intraperitoneal injection of uranyl nitrate at acute doses either nephrotoxic (576 microg of U/kg body weight) or subnephrotoxic (144 microg U/kg body weight). The health effects of U administration at 576 microg of U/kg body weight were seen in terms of decrease in food intake and no gain in body weight compared to respective controls. These alterations were correlated with increased lipid peroxidation as measured by thiobarbituric acid reactive substances in rat brain and bone. However, at lower dosage of U (144 microg U/kg body weight), no significant lipid peroxidation was observed in brain and bone. Histological examination of U-treated (576 microg of U/kg body weight) rat brain tissues showed marked and diffuse cystic degeneration and a similar pattern in histological alterations was observed in kidneys in treated animals; whereas no significant histological change was observed in rat brains and kidney treated with a lower dose of U (144 microg U/kg body weight). It is concluded that administration of U at an acute nephrotoxic dose caused oxidative stress in brain and bone manifested as lipid peroxidation and histopathological damage.

Altered intravenous pharmacokinetics of topotecan in rats with acute renal failure (ARF) induced by uranyl nitrate: do adenosine A1 antagonists (selective/non-selective) normalize the altered topotecan kinetics in ARF?

A series of exploratory investigations with multiple agents was carried out in normal rats and in rats with uranyl nitrate-induced acute renal failure to understand the disposition characteristics of intravenous topotecan (TPT) used as a model substrate. The disposition of TPT was unaltered in normal rats when treated with methotrexate, whereas treatment with probenecid increased the systemic exposure of TPT. In case of uranyl nitrate-induced acute renal failure (UN-ARF) rats, the systemic exposure of TPT was increased when compared with normal rats, whereas in UN-ARF rats treated with probenecid a further reduction in renal clearance of TPT was noted as compared with that of UN-ARF induced rats. Thus, TPT may be involved in the tubular secretory pathway when a passive glomerular filtration pathway for elimination was not possible. The disposition of TPT did not normalize in UN-ARF rats when treated with caffeine, a non-selective adenosine A1 receptor antagonist, whereas the selective adenosine A1 receptor antagonist (1,3-dipropyl-8-phenylxanthine, DPPX) normalized TPT pharmacokinetic disposition by improving renal function. Renal excretion studies demonstrated that CLR improved by almost fivefold following DPPX treatment in ARF rats. In addition, the qualitative stability/metabolism pattern of TPT in liver microsomes prepared from various groups of rats (normal rats, UN-ARF rats, rats treated with DPPX, and UN-ARF rats treated with DPPX) was found to be similar. In summary, using a pharmacokinetic tool as a surrogate, it has been shown that the pharmacokinetic disposition of TPT improved considerably upon treatment with DPPX, a selective adenosine A1 antagonist.

Comprehensive analysis of the renal transcriptional response to acute uranyl nitrate exposure.

BACKGROUND: Chemical and radiological toxicities related to uranium acute exposure have been widely studied in nuclear fuel workers and military personnel. It is well known that uranyl nitrate induces acute renal failure (ARF). However, the mechanisms of this metal-induced injury are not well defined at the molecular level. RESULTS: Renal function and histology were assessed in mice receiving uranyl nitrate (UN(+)) and controls (UN(-)). To identify the genomic response to uranium exposure, serial analysis gene expression (SAGE) of the kidney was performed in both groups. Over 43,000 mRNA SAGE tags were sequenced. A selection of the differentially expressed transcripts was confirmed by real-time quantitative PCR and Western blotting. UN(+) animals developed renal failure and displayed the characteristic histological lesions of UN nephropathy. Of the >14,500 unique tags identified in both libraries, 224 had a modified expression level; they are known to participate in inflammation, ion transport, signal transduction, oxidative stress, apoptosis, metabolism, and catabolism. Several genes that were identified had not previously been evaluated within the context of toxic ARF such as translationally controlled tumor protein, insulin like growth factor binding protein 7 and ribosomal protein S29, all apoptosis related genes. CONCLUSION: We report a comprehensive description of the UN induced modifications in gene expression levels, including the identification of genes previously unrelated to ARF. The study of these genes and the metabolisms they control should improve our understanding of toxic ARF and enlighten on the molecular targets for potential therapeutic interventions.

In vitro immune toxicity of depleted uranium: effects on murine macrophages, CD4+ T cells, and gene expression profiles.

Depleted uranium (DU) is a by-product of the uranium enrichment process and shares chemical properties with natural and enriched uranium. To investigate the toxic effects of environmental DU exposure on the immune system, we examined the influences of DU (in the form of uranyl nitrate) on viability and immune function as well as cytokine gene expression in murine peritoneal macrophages and splenic CD4+ T cells. Macrophages and CD4+ T cells were exposed to various concentrations of DU, and cell death via apoptosis and necrosis was analyzed using annexin-V/propidium iodide assay. DU cytotoxicity in both cell types was concentration dependent, with macrophage apoptosis and necrosis occurring within 24 hr at 100 microM DU exposure, whereas CD4+ T cells underwent cell death at 500 microM DU exposure. Noncytotoxic concentrations for macrophages and CD4+ T cells were determined as 50 and 100 microM, respectively. Lymphoproliferation analysis indicated that macrophage accessory cell function was altered with 200 microM DU after exposure times as short as 2 hr. Microarray and real-time reverse-transcriptase polymerase chain reaction analyses revealed that DU alters gene expression patterns in both cell types. The most differentially expressed genes were related to signal transduction, such as c-jun, NF- kappa Bp65, neurotrophic factors (e.g., Mdk), chemokine and chemokine receptors (e.g., TECK/CCL25), and interleukins such as IL-10 and IL-5, indicating a possible involvement of DU in cancer development, autoimmune diseases, and T helper 2 polarization of T cells. The results are a first step in identifying molecular targets for the toxicity of DU and the elucidation of the molecular mechanisms for the immune modulation ability of DU.

Fetal nephrotoxicity.

Investigation of fetal nephrotoxicity by maternally administered nephrotoxins is hampered by many constraints, including the maternal effects of the nephrotoxin, the ability of the nephrotoxin to cross the placenta and the difficulties associated with direct fetal intervention. In the pouch young of the North American opossum, Didelphis virginiana, we describe the toxic effects of a heavy metal on the immature metanephric kidneys. Varying doses of uranyl nitrate, a heavy metal salt, were administered to opossum pups in the pouch approximately 20 days after birth and the kidneys were harvested 3 to 12 days later for histological analysis. Group 1 consisted of 4 untreated and 5 saline treated pups. Group 2 (9 pups) received 10 to 15 mg./kg. intraperitoneal uranyl nitrate. Group 3 (6 pups) were given a uranyl nitrate dose of 25 mg./kg. Group 4, the high dose group, received either 58 mg./kg. (3 pups) or 87 mg./kg. (3 pups) of intraperitoneal uranyl nitrate. Group 1 kidneys demonstrated no pathological changes except for some mild renal tubular vacuolization seen in the saline treated animals. In group 2 tubular dilatation and necrosis were present 3 days after treatment; tubular regeneration could be seen by day 7. In group 3 glomerular cystic changes, interstitial fibrosis and tubular regeneration were present by day 7. Some restoration of normal architecture occurred by day 12 with fibrosis apparent. Group 4 animals demonstrated much more pronounced cystic changes of glomeruli and tubules as early as day 5 with marked interstitial fibrosis and prominent tubular regeneration. By day 12 group 4 pups continued to demonstrate significant and severe glomerular and tubular cystic changes with marked interstitial fibrosis. Inflammation, although present in all groups (except control), was never prominent. This first description of the effect of heavy metal toxicity on the immature metanephric kidney could provide an insight into the mechanisms of disordered kidney growth.

Early effects of uranyl nitrate on respiration and K+ transport in rabbit proximal tubule.

The mechanisms by which uranyl nitrate (UN) is toxic to the proximal tubule are incompletely understood. To define these further we studied potassium (K+) transport and oxygen consumption (QO2) in rabbit proximal tubule suspensions in vitro immediately after exposure to UN using extracellular O2- and K+-sensitive electrodes. UN caused a cumulative dose-dependent inhibition of proximal tubule QO2, with a threshold concentration of 5 x 10(-5) M. Kinetic analysis suggested two patterns of cell injury: a higher affinity inhibition of QO2 with a Ki of 5 x 10(-4) M, and a lower affinity inhibition of QO2 with a Ki of 10 mM. QO2 was studied in detail in the presence of these Ki concentrations of UN to define the initial cellular events. The results indicated that different cellular processes displayed different sensitivities to UN. At submillimolar concentrations UN caused progressive selective inhibition of ouabain-insensitive QO2 (15% inhibition at 2 minutes). Ouabain-sensitive QO2 and nystatin-stimulated QO2 were not affected, suggesting that Na+,K+-ATPase activity and its coupling to mitochondrial ATP synthesis were intact. Direct measurement of proximal tubule net K+ flux confirmed that Na+,K+-ATPase activity was unchanged. Similarly, UN did not inhibit basal (state 4) or ADP-stimulated (state 3) mitochondrial QO2 in digitonin-permeabilized tubules, confirming that the mitochondria were intact. In contrast, higher concentrations of UN (greater than or equal to 1 mM) caused rapid inhibition of QO2 and net K+ efflux, due to inhibition of Na+,K+-ATPase activity and mitochondrial injury.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of the transient depression of the erythropoietic rate induced in the rat by a single injection of uranyl nitrate.

With the purpose of assessing the effect of uranyl nitrate (UN) on the rate of erythropoiesis, 1 mg/kg of the compound was injected iv to adult female Wistar rats. The dosing vehicle was injected into control animals. A single injection of UN induced a transient depression of the rate of red cell volume 59Fe uptake, which reached its lowest value (68% depression) by the seventh postinjection day. By 14 days, 59Fe incorporation had returned to normal. The amount of iron going to erythroid tissue per hour, reticulocyte count, and immunoreactive erythropoietin concentration in both plasma and kidney extracts were also significantly depressed in UN-treated rats in relation to these values in vehicle-injected rats by the seventh postinjection day. Dose-response curves for exogenous erythropoietin (Epo) performed in polycythemic intact and UN-treated rats 7 days after drug injection revealed a significant depression of the response in UN-injected animals. Moreover, bone marrow cells obtained from rats pretreated with UN formed a reduced number of erythroid colonies in vitro in response to Epo. Therefore, possible mechanisms for the observed transient depression in the rate of erythropoiesis associated with acute UN treatment include decreased Epo production and direct or indirect damage of erythroid progenitor cells.

Transport of p-aminohippurate, tetraethylammonium and D-glucose in renal brush border membranes from rats with acute renal failure.

Transport of D-glucose, p-aminohippurate and tetraethylammonium has been studied using renal brush border membrane vesicles isolated from rats with uranyl nitrate-induced acute renal failure (ARF). Initial rate and overshoot magnitude of Na+ gradient-dependent D-glucose uptake were decreased in brush border membrane vesicles from ARF rats compared with normal rats, although there was no significant difference on D-glucose uptake in the presence of equilibrated Na+ between normal and ARF rats. Uptake of p-aminohippurate by membrane vesicles from ARF rats did not differ from normal membrane vesicles. Uptake of tetraethylammonium with or without an H+ gradient was decreased in membrane vesicles from ARF rats compared with normal rats. Dissipation rate of H+ gradient across brush border membranes did not differ between both groups. In vitro incubation of normal brush border membrane vesicles with uranyl nitrate caused no alteration in any substrate transport. However, enzyme activities such as (Na+ + K+)-adenosine triphosphatase in renal cortical homogenate were inhibited markedly in the presence of uranyl nitrate. These results suggest that uranyl nitrate-induced ARF caused alterations in the transport properties of renal brush border membranes and that these transport dysfunctions were not due to the direct effect of uranyl nitrate, but could be secondarily induced after the impairment of the integrity for tubular cells.