| Lead (Pb) and cadmium (Cd) are now recognized to be two of most important heavy metal contaminants in the environment. Due to their increased industrial uses and environmental pollution with the related waste products, concentrations of lead and cadmium are increasing rapidly in the environment in recent years. Since the two elements are often released simultaneously in the environment from a number of natural and man made sources, adverse health effects caused by combined exposure to lead and cadmium has provoked a significant public health concern. The kidney is the target organ and the primary accumulation site of chronic lead and cadmium exposure. The nephrotoxicity induced by lead and/or cadmium have been extensively studied and widely reported in occupationally and environmentally exposed human subjects, as well as in various experimental models. Most studies were implicated in the single exposure of lead/cadmium on the kidney. However, systemic studies of toxic damage on the combination of lead and cadmium were little referred. In this study, the toxic effects of lead and/or cadmium on the kidney of Sprague-Dawley (SD) rats were investigated in vitro and in vivo, which will offer some theoretic evidences for further exploring the mechanism in nephrotoxicity of lead and/or cadmium.1. In vivo studies The study was carried out on female one-month-old SD rats. Twenty- four rats were allocated randomly to four groups of six animals each. The experimental period was eight weeks. (1) Control: rats consumed distilled water as drinking water. (2) Lead treated group: rats consumed a solution of PbAc2 (300mg/L) as drinking water. (3) Cadmium treated group: rats consumed a solution of CdAc2 (50mg/L) as drinking water. (4) Pb+Cd treated group: these rats received both Pb and Cd at the doses, periods and ways of administration described above. During the experimental period, water consumption and weight gain were measured every day. A series of tests were carried out:①On the day before the experiment and at the end of 2, 4, 6 and 8 weeks of treatment, rats were kept for 24h urine collection. Activities of alkaline phosphatase (ALP), N-acetyl-β-D-glucosaminidase (NAG),γ-glutamyl-transpeptidase (GGT), lacticacid dehydrogenase (LDH) and contents of total protein (TP),α1-microglobulin (α1-MG),β2-microglobulin (β2-MG), microalbumen (mAlb) in urine were determined. Also, concentrations of Zn, Cu, Mn, Fe and Se in the urine were detected during the experiment.②At the end of treatment, the levels of Zn, Cu, Mn, Fe, Se, glutathione (GSH), malondialdehyde (MDA) and activities of total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) in the renal cortex and serum were measured.③Changes in histopathology and ultrastructure of renal cortex were detected by light microscope and transmission electron-microscope, respectively.④The relative gene expression levels of cytochrome oxidase submits (COX-I, COX-II, COX-III) in the renal cortex were quantified by Fluorescent Quantitative-PCR (FQ-PCR). The expressions of metallothionein submits (MT-1, MT-2) in the renal cortex were detected by immunohistochemistry method and FQ-PCR, respectively. The results are as follows:①Compared with the control group, all of the indices(NAG, GGT, LDH, TP,β2-MG, mAlb) except the contents ofα1-MG in the Pb, Cd group and activities of ALP in the Pb group at the time of 2-week exposure increased significantly from the beginning of exposed for two weeks (P<0.05 or P<0.01). Especially the changes in the (Pb+Cd) group were the greatest (P<0.05). Positive correlation lied in the process between the degree of increase in activities of urinary enzymes/contents of urinary proteins in the exposed groups and the exposure time. During the experiment, the levels of indices in the (Pb+Cd) group were all higher than those in the Pb, Cd group. There is significant difference between the (Pb+Cd) group and Pb or Cd group in different times.②In comparison with the control group, activities of SOD, CAT, GSH-Px and contents of GSH, Zn, Cu, Mn, Fe, Se in the renal cortex and serum of the three exposed groups decreased significantly(P<0.05). However, the contents of MDA in these exposed groups were significantly increased than that of the control group (P<0.05).③As far as the Pb group and the Cd group is concerned, concentrations of trace elements (Cu, Mn, Fe, Se) in the urine except the concentrations of urinary Zn at the time of 2-week exposure increased significantly from the beginning of exposed for four weeks(P<0.05). Regarding the (Pb+Cd) group, the excretion of trace elements (Zn, Cu, Mn, Se) in the urine except that of urinary Fe increased significantly from the beginning of exposed for two weeks (P<0.05).④Obvious pathological changes in the renal tubule and glomeruli renis were observed in the renal cortex of these three exposed groups. Also, the changes in ultrastructure of renal cortex is obvious, which pyknosis of nucleus, chromatin assemble, partial loss of brush border microvilli in the proximal tubular cells, mitochondrial swelling, disappearance and fragmentation of carina were seen under transmission electronic microscope. The degree of pathological damage in the (Pb+Cd) group was more serious than that in the Pb/Cd group. Compared with the Pb or Cd group, more severe pathological damage was found in the (Pb+Cd) group.⑤The relative expression levels of cytochrome oxidase submits (COX-I, COX-II, COX-III) in the renal cortex of these three exposed groups were significantly lower than those in the control group (P<0.05). The most significant change occurred in the (Pb+Cd) group.⑥There is no significant difference in the expression level of MT-1 and MT-2 gene in the kidneys between the lead group and control group (P>0.05), whereas those in the cadmium group and (Pb+Cd) group were significantly higher than that in the control group (P<0.05). Based on these results, the conclusions are as following:①Tubular reabsorptive function and glomerular filtration function were damaged after exposed to lead and/or cadmium. The degree of renal injury is positively correlated with the increase of exposure time.②Exposure to lead and/or cadmium can induce the decrease of the anti-oxidative function in the kidneys of rats. Also the decreased contents of trace elements related to antioxidative function made the renal damage induced by oxidative stress worse. The decreased levels of these trace elements in the tissues exposed to lead and/or cadmium were due to an increase of their excretion in the urine.③Obvious pathological changes and damage of many organelles in the renal cortex were medicated by lead and/or cadmium. Among these organelles, mitochondria underwent the greatest changes.④The decreased expression levels of COX-I, COX-II, COX-III in the renal cortex exposed to lead and/or cadmium may be related to the mitochondrial lipid peroxidation. The increased expression level of MT-1 and MT-2 gene played an important role in the nephrotoxicity induced by lead and cadmium. In summary, there was an obvious synergistic effect of lead combined with cadmium on the kidney of rats.2. In vitro studies The primary cultures of rat proximal tubular cells (rTECs) were cultured by mechanical grinding, filtering and chemical digestive methods. The first passage was used to perform the experimental design when it was in its highest cell viability. Effects of lead (0.5μmol/L, 1μmol/L) and/or cadmium (2.5μmol/L, 5μmol/L) on the rTECs were investigated in the following assays.①Effects of different doses of lead and/or cadmium on the survival rates in rPTCs for a time range of 3, 6, 12 and 24h were detected by using the cck-8 reduction method.②Effects of lead and/or cadmium on the apoptotic rates, necrotic rates, LDH release and apoptotic morphological changes in rPTCs over a 12 h period were investigated. In addition, the protective effect of N-acetyl-L-cysteine (NAC) against lead and/or cadmium induced cellular damage was investigated.③Activities of SOD, CAT, GSH-Px and contents of GSH, MDA in rPTCs were measured when exposed to lead and/or cadmium over a 12h period.④Activities of Ca2+-ATPase and Na+/K+-ATPase, intracellular pH, levels of mitochondrial membrane potential (ΔΨ), reactive oxygen species (ROS) and intracellular [Ca2+]i in rPTCs were detected after exposed to lead and/or cadmium for 12h. The results are as follows:①The cell survival rates in the single lead and cadmium groups were significantly lower than those of control groups since these cells were exposed to high-dose and low-dose groups for three and six hours, respectively (P<0.05). The cell survival rates in the combined groups were significantly lower than those of control groups after a 3-h exposure (P<0.01). Furthermore, the degree of decrease in the cell survival rate was positively correlated with the dose and the exposure time.②After exposure to lead and/or cadmium for 12h, the apoptotic rates, necrotic rates, LDH release in these exposed groups were significantly higher than those in the control group (P<0.01). Also, the above indices induced by (Pb+Cd) were always higher than those in the related Pb or Cd group in the same exposure time. After a 12-h exposure time, it showed morphological changes typical of apoptosis in the lead and/or cadmium groups, i.e., nuclear chromatin condensed and fragmented chromatin was characterized by a scattered, drop-like structure. Apoptosis induced by lead and/or cadmium can be efficiently prevented by NAC, but the necrotic rates and LDH release were not affected by NAC.③Compared with the control group, activities of SOD, CAT, GSH-Px and the GSH level in the exposed groups decreased significantly (P<0.05 or P<0.01); But the content of MDA increased significantly (P<0.01).④After exposed to lead and/or cadmium for 12h, intracellular ROS and [Ca2+]i in rPTCs increased significantly (P<0.01), while the mitochondrialΔΨ, intracellular pH, activities of Ca2+-ATPase and Na+/K+-ATPase decreased significantly (P<0.05 or P<0.01). Based on these results, the conclusions are as follows:①Lead and/or cadmium exposure induced cellular death in rPTCs, depending on both the concentration and the exposure time. Synergistic effect lies in the administration of lead combined with cadmium.②Cellular death induced by lead and/or cadmium is medicated by two mechanisms, necrotic and apoptotic. The apoptotic mechanism played a chief role in the cellular death induced by lead and/or cadmium at these doses. Moreover, oxidative stress could be implicated in the apoptotic mechanism mediated by lead and/or cadmium. Decreased activities of anti-oxidative enzymes further enhanced oxidative damage in rPTCs caused by lead and/or cadmium. In addition, the cellular damage induced by lead and/or cadmium can be significantly prevented by NAC.③Depletion of mitochondrialΔΨand a disorder of intracellular homeostasis, i.e. intracellular acidification, calcium overload, disturbance in the prooxidant–antioxidant balance, promoted the development of apoptosis in rPTCs. In a word, there was an obvious synergistic effect of lead combined with cadmium on the cellular damage in rPTCs.
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