Study On The Effects Of Cadmium On E-cadherin In The Proximal Tubule And The Effects Of Chlorpromazine And Verapamil On Cadmium Toxicity

ObjectiveCadmium (Cd) is an important industrial and environmental pollutant that poses a significant health risk to humans and animals. Depending on the dose, route, and duration of exposure, Cd can cause damage to various organs including the lung, liver, bone and kidney. In addition, Cd has been shown to have teratogenic and carcinogenic activities. Kidney is the major target organ of Cd -induced chronic renal damage. Due to its accumulations in the kidney, it causes tubular dysfunctions and, in many cases, chronic damage. While the general effects of Cd on renal function have been fairly well characterized, relatively little is known regarding the specific mechanisms by which Cd actually damages kidney. Cd can produce a wide variety of cellular effects, including inducing ox-idative stress, interfering with calcium homeostasis, and interfering with the normal function of the Ca+ - dependent cell adhesion molecule E - cadherin ( E -cad). However, the recent studies show that E - cad may be a relatively early target of Cd toxicity. And the Cd - induced damage of E - cad - dependent adhesion junction may contribute to Cd renal toxicity.The aim of the present study is to research the effects of Cd on E - cad in the proximal tubule cultured primarily and the relationship between E - cad damage and other cell damage; According study in vitro, a subchronic Cd exposure protocol is established to induce renal dysfunction in rats to study further the effect of Cd on E - cad in the proximal tubule in vivo and the relationship between E - cad damage and other damage; Treating rats injected with CdCl2with either CaM antagonist CPZ or Ca2 + antagonist Ver is to research the effects of CPZ or Ver on Cd toxicity. Those offer the important data for further discussing the molecular mechanisms of the toxicity induced by Cd.Methods1. Effects of cadmium on E - cadherin in the proximal tubule in vitro The proximal tubules were isolated by screen separation method from 5d -old newborn Wistar rats. The abdominal cavity was opened in asepsis worktable and kidney was exposed. Kidney was quickly removed and renal cortex was cut free in DMEM/F12 medium. Renal cortex was cut into lmm3 small pieces, which was washed in DMEM/F12 medium. Small pieces were skived on 80 - order screen with grinder. Liquid was filtrated with 100 - order screen. Tubule segments were collected on the 100 -order screen. Then, tubule segments were treated by 0. 2% trypsinization 2 -3ml for 30 min at 37°C in couveuse. Adding DMEM/F12 medium with 15% fetal bovine serum (FBS) was in order to stop digestion. The liquid containing tubule segments was centrifuged for 5 min. The supernatant was discarded. Isolated cell and tubule segments were platede in DMEM/F12 medium with 15% FBS. The cells were 0 -4h exposed to different levels of CdCl2 (10, 20, 50 and 100jxmol/L). The variety of cell shape was observed. Cell viability, LDH activities in medium and ALP activities in cell were determined as well as GSH contents and the levels of Ca2 +. It was mensurated that the effects of Cd on the localization of E - cad by immunohistochemistry.2. Effects of cadmium on E - cadherin in the proximal tubule in vivo Wistar rats weighing 110 ± lOg were obtained from Laboratory Animal Center in China Medical University. The quantity of male was the same as that of female. The animals were housed at 17 ~23Tl. Rat chow was provided by Laboratory Animal Center. Animals were fed for seven days before experiment, then they were divided randomly into two groups: the control and the Cd groups. Three days each week, the animals in the Cd treatment group ( n = 6) received subcutaneous injections of CdCl2 at a Cd dose of 1. 4mg/kg in isotonic saline o-ver a total period of 4 weeks. The animals in the control group (n =6) receivedinjections of the saline alone. On the end of the fourth week, the animals were placed in individual metabolic cages, and 24 h urinary samples were collected. Then the animals were anesthetized with ethyl ether. The abdominal cavity was opened and kidney was exposed. Kidney was quickly removed and portions of the renal cortex were cut free. It was determined that urinary LDH activities, u-rinary ALP activities, urinary protein, GSH, MDA contents and Ca2 + concentrations in renal cortex. Imrnunohistochernistry and in situ hybridization of E - cad were determined.3. Effects of CPZ and Ver on cadmium toxicity3. 1 Effects of CPZ and Ver on liver and kidney of rats with acute exposure to cadmiumForty male and female Wistar rats, 140 ± 10 g of weight, were purchased from Laboratory Animal Center in China Medical University. The animals were housed at 17 ~23Xl. Rat chow was provided by Laboratory Animal Center. The animals were fed for seven days before experiment, and then they were divided randomly into four groups: the control group and three Cd groups. Three Cd groups were injected subcutaneously with 25fxmol/kg CdCl2 CPZ or Ver treated groups respectiviely received ip injection of 12.5mg/kg CPZ or lOmg/kg Ver lh before 25p,mol/kg CdCl2 injection. The animals in the control group received injections of the saline alone. After 24h, the animals were anesthetized with ethyl ether. Blood samples were collected with puncturing into abdominal aorta. All the animals were killed, and the liver and kidney were collected. Renal cortex was cut free. LDH and GPT activities in serum were measured. liver and renal cortex were used to measure Cd contents, MDA contents, GSH contents, and GSH - Px activities.3.2 Effects of Ver and CPZ on cadmium - induced nephrotoxicityThirty - two Wistar rats, 110 ± lOg of weight, were purchased from Laboratory Animal Center in China Medical University. The quantity of male was the same as that of female. The animals were housed at 17 ~23T!. The rat chow was provided by Laboratory Animal Center. The animals were fed for seven days before experiment, then they were divided randomly into four groups: the control group and three Cd groups. Three Cd groups were injected subcutaneously withCdCl2containing Cd 1.4mg/kg 3 days per week for up to 6 weeks. Two of three Cd groups received ip injection of 4mg/kg Ver or 5mg/kg CPZ lh before CdCl2 injection. The animals in the control group received injections of the saline a-lone. At the end of the 2, 4 and 6 weeks, the animals were placed in individual metabolic cages, and 24h urinary samples were collected. After 6 week, the animals were anesthetized with ethyl ether. Blood samples were collected with puncturing into abdominal aorta. All the animals were killed, and kidney was collected. Renal cortex was cut free. Urinary NAG activities and urinary protein were measured. BUN in serum was determined. Cd and Ca contens were measured in renal cortex, blood and urine. MDA contents were measured in renal cortex and blood.4. Statistic analysisAll results were expressed as mean ± standard deviation and treated in SPSS12. 0 software by one -way analysis of variance (ANOVA) to test the significant differences among groups followed by q test ( Student - Newman -Keuls, SNK) to find the differences between the two groups, t test was used to determine the levels of significance between two independent samples.Results1. Effects of cadmium on E — cadherin in the proximal tubule in vitro Cells were beginning to detach from each other after lOmin - exposure to lOjxmol/L CdCl2. During the course of 30min - lh - exposure to lOjxmol/L CdCl2, light around most of cells increased significantly. For 4h - exposure to lOOfimol/L CdCl2 decreased obviously cell viability as compared the control. For 4h - exposure to lOOjxmol/L CdCl2 as compared 2h - exposure, cell viability showed obvious change. For different time (4h, 2h, lh and 0.5h) exposure to different levels of CdCl2(10, 20, 50 and lOOjxmol/L) significantly reduced the ALP activities, which presented certain dose - time effect relationship. For 4h, 2h - exposure to 50 and 100|xmol/L CdCl2 increased LDH activities in media as compared the control. For 4h - exposure to 50jxmol/L CdCl2 decreased GSH content. GSH content for 2h - exposure to lOOjxmol/L CdCl2 was 15% ofthat in the control, and 8. 3% for 4h - exposure. For 4h - exposure to 50 and 100fimol/L CdCl2 decreased significantly the levels of intracellular Ca2 + . E -cad expression was decreased after 2h - exposure to 10fimol/L CdCl2. Almost no E - cad was expressed in cytoplasm after lh - exposure to 100u>mol/L CdCl2.2. Effects of cadmium on E - cadherin in the proximal tubule in vivo The Cd - treated animals gained significantly less weight than did the control animals over the 4 - week course of treatment. The Cd - treated animals did show a significant increase in urinary LDH, urinary ALP activities and protein contents. The samples from the Cd - treated animals showed a significant increase in the levels of Ca2+ in renal cortex as compared those from the control group. The samples from the Cd - treated animals showed only a very slight increase in the levels of GSH and no significant change in the levels of MDA. The proximal tubule in the samples from the control animals exhibited regular cubical shapes, well - defined nuclei, and a full cytoplasm, and they were closely associated with adjacent cells. By contrast, the proximal tubule in the samples from the Cd - treated animals showed a very ragged, irregular appearance, with cells swelling and smaller lacuna. A massive brush border appeared to falling off, with partial vacuolede naturalization. But the nuclei of the cells in the sections from the Cd - treated animals appeared to be normal in size, shape, staining density, and pattern of orientation. E - cad was largely present in the basolater-al surface in the control samples. It also appeared in cytoplasm. In addition, it appeared a clear band of labeling at apical cell surface in some of the proximal tubules. But the samples from the Cd - treated group showed most of the tubules showed a blurrily band of labeling in the basolateral surface. The basolateral labeling of E - cad was less localized and more diffuse than in the control samples. The number of E - cad in cytoplasm relatively increases. Results in situ hybridization showed E - cad was largely present in cytoplasm both in the control samples and in the Cd - treatment samples and the location of E - cad was not changed. The samples of immunohistochemistry from the Cd - treated animals consistently showed statistically significant reduction in the scores for integrated OD total and optical density average of E -cad labeling. By contrast, the sam-pies of in situ hybridization from the Cd - treated animals didnt show the significant diffidence in the scores for integrated OD total and optical density average of E - cad labeling as compared these from the control animals. 3. Effects of CPZ and Ver on cadmium toxicity3.1 Effects of CPZ and Ver on liver and kidney of rats with acute exposure to cadmiumLDH activities and GPT activities in serum in the Cd - treated animals increased significantly as compared the control group indicating liver was damaged. Cd obviously increased the levels of MDA and GSH in liver, and decreased GSH -Px activities in liver, indicating liver was under oxidative stress. CPZ decreased obviously LDH activities in serum, and to some extension decreased GPT activities in serum and the contents of Cd in liver. CPZ made MDA reduce 50% of that in the control, and decreased GSH in liver acute 24h - exposure to Cd. Those indicated that CPZ lighted oxi dative stress in liver induced by Cd. Ver decreased significantly LDH activities and GPT activities in serum, especially reduced 61. 4% GPT activities as compared that in the Cd - treated group. Ver also decreased significantly the levels of MDA and GSH in liver, increased GSH - Px activities in liver. Those suggested Ver lightened oxidative stress in liver induced by acute exposure to Cd. CPZ increased significantly GSH contents in renal cortex, and Ver increased significantly GSH - Px activities in renal cortex.3.2 Effects of Ver and CPZ on cadmium - induced nephrotoxicity Urinary NAG activities and urinary protein in the Cd - treated group increased significantly as compared the control group at the 4 week. Cd contents and Ca contents in blood, renal cortex and urine in the Cd - treated group showed significant change as compared the control group at the 6 week, indicating that rats'kidney was damaged. At the 4 week, Ver decreased significantly urinary NAG activities and urinary protein. At the 6 week, Ver decreased significantly BUN in serum and Cd contents in blood, and decreased 62. 8% Ca contents in urine. Those indicated Ver postponed and lighted Cd - induced renal damage. At the 6 week, Ver .decreased significantly 58.6% MDA in renal cortex indicating Ver lighted renal oxidative stress induced by Cd. Urinary NAG ac-...