Effect Of Depleted Uranium On The Kidney And Immune System And The Protective Role Of Zinc

Background and ObjectivesDepleted uranium (DU) is the by-product of uranium (235U) enrichment from naturaluranium, as having235U content lower than0.712%. Due to its efficient penetration andaffordability, DU has recently been widely used in nuclear power plants, counterweights,radiation protection and military activities (such as armour material and ammunitioncomponents). However, during its production and use, DU may be released into theenvironment due to failure to follow standard procedures, thus causing environmentalpollution. DU may eventually enter the human body through the respiratory tract, digestivetract or skin, leading to contamination of local residents.DU emits α and β particles with high linear energy transfer, and DU has the dualeffects of radioactive toxicity and heavy metal toxicity. The biological effects of DU areaffected by the exposure dose, exposure duration, the exposure pathway and many otherfactors. During acute high-dose exposures, the kidney is the main target organ of thechemical toxicity of DU, which may cause severe tubular necrosis. However, themechanism of DU-induced nephrotoxicity is not clear. Low-dose chronic exposure maycause a series of harmful effects, such as neurobehavioral abnormalities, genetic toxicity,reproductive toxicity, and cancer. However, few published studies exist on the impact ofDU on immune function in live animals. There is still no report on the effects of ingestingDU-contaminated food on the immune system, which continues to be a worldwide concern.Therefore, the present study created an acute toxicity model and a chronic toxicity modelthrough long-term ingestion of DU-containing feed. We evaluated the effect of DU on thekidney after acute exposure and the immune system after chronic exposure. We alsoexplored in depth the mechanism of DU-induced toxicity. This study was a basic for thestudy of the biological effects of DU.In addition, the prevention and treatment of DU intoxication is still a worldwide keyand difficult point. However, at present, most of chelating agents for DU still have someinsufficiencies more or less in removal effect or security. MT is a low-molecular weight, thiol-containing protein that is found throughout the body and that participates inscavenging free radical and heavy metal detoxification. However, the mechanism involvedin the protective role of MT remains an enigma. The gene expression of MT is dependenton the zinc ions. Zinc is an efficient and safe inductor of MT. Therefore, we used zinc todetoxify the DU intoxication by inducing MT. This study provided a new idea for thedevelopment of the high-performance and low-toxicity drugs against DU in clinicapplications, and might have an important practical value.MethodsFirst, human kidney cells (HK-2) and Sprague Dawley rats were used to expose to thedifferent doses of DU for creating an acute toxicity model, respectively. After24h exposureto DU, the cell viability, apoptosis and related enzyme activity were assessed in HK-2cells,and after4d exposure to DU, kidney uranium content, renal function, histopathology andantioxidant levels in tissue were measured in rats.Second, three-week-old Kunming mice were divided into the following4groups basedon the various feeding doses (containing DU):0(control group),3(DU3group),30(DU30group), and300mg/kg feed (DU300group). After4months of exposure, the innate immunefunction and acquired immune function were evaluated.Third, HK-2cells, Sprague Dawley rats and MT knockout mice were used to study theprotective role of zinc against DU, respectively. The methods including histomorphology,ultrastructural pathology, flow cytometry, laser scanning confocal microscope, enzymelinked immunosorbent assay (ELISA), molecular biology and proteomics were used toevaluated the detoxification of zinc against DU. The mechanism of protective role of zincagainst DU was explored in depth.Results and Conclusion1. After24h exposure to uranyl nitrate (more than125μM), cytoplasmic membranesand lysosomal membranes of HK-2cells damaged, and lactate dehydrogenase (LDH) andN-acethyl-β-D-glucosaminidase (NAG) were released from cell to culture medium, leadingto cell apoptosis at last. In addition, Sprague Dawley rats were exposed to different doses ofDU (2.5、5、10mg/kg). After4d exposure, DU (more than2.5mg/kg) led to obviousnephrotoxicity, including increasing kidney uranium content, blood urea nitrogen (BUN)and creatinine (Cr) levels, and serious pathological damage. The nephrotoxicity becamemore severe with higher DU dose. Further studies showed that the antioxidant function decreased and lipid peroxidation level increased in the kidney tissue after exposure to DU,which may be one of the mechanisms of DU-induced nephrotoxicity.2. Three-week-old mice were fed with various doses of DU for4months. After4months of exposure, the biochemical parameters in blood serum of each group were all innormal level. In the DU30and DU3groups, immunological changes were either minor orindiscernible. However, in the DU300group, the innate immune function decreased,manifesting as decreased secretion of nitric oxide, interleukin (IL)-1, IL-18, and tumournecrosis factor (TNF)-α in the peritoneal macrophages, as well as reduced cytotoxicity ofthe splenic natural killer cells. Moreover, the cellular and humoral immune functions wereabnormal, as manifested by decreased proliferation of the splenic T cells, proportion of thecluster of differentiation (CD)3+cells, ratio of CD4+/CD8+cells and delayed-typehypersensitivity, and increased proliferation of the splenic B cells, total serumimmunoglobin (Ig) G and IgE, and proportion of splenic mIgM+mIgD+cells. Throughstimulation, the secretion levels of interferon (IFN)-and TNF-α in the splenic cellsreduced, and the levels of IL-4and IL-10increased. In conclusions, chronic intake ofhigher doses of DU (300mg/kg) had a significant impact on the immune function, mostlikely due to an imbalance in T helper (Th)1and Th2cytokines.3. Mitochondrial and FasR-mediated apoptosis pathways contribute to DU-inducedapoptosis in HK-2cells. Pre-treatment with zinc (100μM) significantly inhibited DU(500μM)-induced HK-2cells apoptosis. It reduced the formation of reactive oxygen speciesin the cells, increased the catalase (CAT) and glutathione (GSH) concentrations, suppressedthe DU-induced soluble Fas receptor (sFasR) and soluble Fas ligand (sFasL)overexpression, suppressed the release of cytochrome c and apoptosis inhibitor factor (AIF)form mitochondria to cytoplasm, inhibited the activation of caspase-9, caspase-8, andcaspase-3, and induced MT expression. Furthermore, exogenous MT effectively inhibitedDU-induced cell apoptosis. In conclusions, through independent mechanisms, such asantioxidant effects, inhibition of the activation of caspase-9, caspase-8, and caspase-3, andinduction of MT expression, zinc inhibits DU-induced apoptosis.4. Pre-treatment with zinc (10mg/kg) had significantly higher survival rates than ratswithout zinc pre-treatment at30d post depleted uranium administration. At4d postadministration, the former had higher urine uranium content but lower liver and kidneyuranium content. Meanwhile, serum BUN and Cr levels and urine NAG concentrations significantly decreased; Gene expression levels of MT-1and MT-2in kidney tissuessignificantly increased; and CAT levels increased and malonaldehyde (MDA) levelsdecreased, both significantly. Compared to rats exposed only to DU, rats with zincpre-treatment had insignificant renal tubular epithelial cell necrosis and less transparenttubes. With regard to mice, after DU injection, both MT+/+and MT-/-mice showedsignificantly increased uranium content of the renal tissues and serum Cr and BUN.Meanwhile, renal tissues displayed severe renal pathological damage. However, thesechanges were more evident in MT-/-mice. Zinc pre-treatment significantly decreaseduranium content in renal tissues with increasing MT content, and alleviated renal damage inMT+/+mice, which was not observed in MT-/-mice. Furthermore, exogenous MT1andMT2all could significantly decrease the kidney uranium content at4d post DUadministration in MT-/-mice. MT1also alleviated renal damage, and increased theantioxidant activity, while MT2could not. In conclusion, MT, not zinc, plays a crucial rolein antagonising the nephrotoxicity of DU. The underlying mechanism includes DUexcretion, which is facilitated by the stable binding of MT to DU in the kidney, and theantioxidant activity of MT, which cleans excess reactive oxygen species and preventssuperoxide generation from the renal tissue, especially MT1.5. An expression proteomic analysis involving two-dimensional electrophoresis andmatrix-assisted laser desorption ionization time-of-flight mass spectrometry was used toidentify proteins changes between MT+/+and MT-/-mice after exposure to DU. Thirteenproteins were identified, whose functions were focus on glycometabolism, fatty acidmetabolism, electron transport respiratory chain and antioxidant defense system. Furtherstudies showed that, the levels of superoxide dismutase (SOD) and amino-acylase3(ACY-3) decreased after exposure to DU. They decreased more significant in MT-/-mice,which suggested these proteins might participate in DU-induced toxicity. Western blot wasused to assess the expression of SOD and ACY-3, and the results were in accord with theproteomic analysis. The results also verified that one of the mechanisms of DU-inducednephrotoxicity was oxidative stress. In addition, ACY-3might be a new target ofDU-induced nephrotoxicity, and MT could alleviate the DU-induced kidney damagethrough modifying SOD and ACY-3expressions.