吸烟相关剂量镉对大鼠肾脏形态和功能影响的实验研究
摘要
目的:观察吸烟相关剂量镉对大鼠肾脏形态结构及功能的影响,探讨吸烟相关剂量镉对肾脏的作用和可能的机制。
方法:建立吸烟相关剂量镉的动物模型,设立模型组和对照组,于20W、28W、36W、44W和52W处死大鼠;应用ICP-MS检测肾脏组织镉、铜、钙、锌元素含量;应用罗氏COBAS Integra 400 Plus全自动生化分析仪检测血清和尿液中BUN、Crea含量及血清钾、钠、钙和氯离子含量;组织切片光镜观察形态学变化;免疫组织化学方法检测MTs及PCNA、AQP-1、AQP-2、AQP-3表达;应用水通透性测定方法检测镉离子对AQP-1的水通透系数;Western blot检测肾脏外髓组织AQP-1蛋白表达。
结果:在吸烟相关剂量镉动物模型组,与对照组比较,各时间点肾脏镉元素含量均有显著差异(P<0.01),并且镉与钙及铜元素之间呈负相关(分别为rs=?0.9,rs=?1.0);与对照组比较,36W时血清BUN、Crea差异明显(P < 0. 05);与对照组比较,尿液肌酐在36W、44W及52W差异显著(P < 0. 05);肾脏组织学观察显示,早期模型组肾脏小管组织结构有一定的破坏,后期则出现上皮不典型增生;吸烟相关剂量镉诱发的MTs定位在肾脏皮质小球、外髓及内髓近曲小管,在细胞胞浆中表达;MTs免疫组化结果表明,模型组20W时表达最强,随模型时间延长逐渐减弱,到52W时仍高于对照组,各时间点模型组与相应对照组比较有显著差异(P<0.01);PCNA在肾小球系膜细胞和小管细胞的表达阳性细胞数呈现先下降随后上升并增多的变化,显示吸烟相关剂量镉干扰了DNA的正常合成;吸烟相关剂量镉模型组肾脏AQP-1、AQP-2及AQP-3蛋白表达降低,随模型时间延长有一定恢复;与对照组比较,直小血管的AQP-1表达差异显著(P<0.01),随模型时间延长逐渐减弱,52W时降至最低;镉对红细胞水通透性没有“门控”作用;Western blot检测模型组肾脏外髓组织AQP-1蛋白表达量下降。
结论:吸烟相关剂量镉可在肾脏持续性累积,造成肾脏功能及形态的损伤,并出现上皮不典型增生,有可能成为肾癌发生的病变基础,其机制可能与镉影响DNA的正常合成有关。该研究提示要重视吸烟相关剂量镉对肾脏的损伤,尤其是不能忽视其远期效应,为说服人们戒烟以减少通过烟草摄入镉提供了理论和实验依据。
The incidence of renal cancer rates second in the urinary system neoplasm, and accounts for 80%-90% of renal malignancies in adults. Renal cancer originates from the epithelia of proximal tubules. Smoking is the main cause of renal cancer, and at least 39% of cases in male are related with it. Cadmium is the major deleterious component in the cigarette and meantime the major pollutant in the environment. The kidneys are the organs impaired most by chronic low-dosage cadmium exposure. Long-term and low-dosage cadmium exposure may impair the epithelia of proximal tubules, resulting in the decreased reabsorption function of proximal tubules. This experiment explored the effect of smoking-dosage cadmium on renal morphology and function.
Objective: to observe the influence of smoking-dosage cadmium on renal morphology and function in rats, and explore the effect of smoking-dosage cadmium on the kidneys and the possible mechanism.
Method: built the animal model of smoking-dosage cadmium; tested the content of cadmium, copper, calcium and zinc in the kidneys with ICP-MS; tested the BUN and Crea content in the serum and urine, as well as the content of potassium, sodium, calcium and chlorine ions in the serum with ROCHE COBAS 400 full-automatic biochemistry analyzer; sliced the tissue and observed the morphologic change under the optic microscope; examined the expression of MT, PCNA, AQP-1, AQP-2 and AQP-3 with immunohistochemical method; measured the water permeability coefficient of cadmium ion to AQP-1 with the water permeability test method; and inspected the protein expression of AQP-1 in the medullary tissue of the kidneys with Western blot.
Results: in the animal model of smoking-dosage cadmium, the coefficients of organs in rats were lower than those of control group(P<0.05)at the 20th week. Compared with the control group, contents of renal cadmium in the model group at various time had significant difference, and cadmium was negatively related with calcium and copper; the serum BUN and Crea had difference at the 36th week compared with the control group; the urine Crea had difference at the 36th, 44th and 52nd week compared with the control group. It was observed under the optic microscope that the tissue structure of renal tubule was impaired in the early phase of the model group, and atypical hyperplasia was observed in the later phase. MTs induced by smoking-dosage cadmium were located in the glomerulus, the outer medullar and proximal tubules in the inner medullar, and expressed in the cell plasma. MTs showed significant difference between the two groups, MTs expression was strongest in the treatment group at the 20th week, and attenuated with the model time prolonging, and it was still higher than the control group at the 52nd week. In the expression of PCNA in the mesangial cells and tubule cells, the positive cell number showed the trend of decreasing first and then increasing, indicating that the smoking-dosage cadmium interfered with the normal synthesis of DNA. Protein expression of AQP-1, AQP-2 and AQP-3 in the kidneys of the smoking-dosage cadmium group decreased, but restored to some extent with the model time prolonging. Protein expression of AQP-1 in the vasa recta had significant difference, attenuated with the model time prolonging and dropped to the lowest at the 52nd week compared with the control group. Cadmium didn’t exert“door control”effect to the water permeability of red cells. Protein expression of AQP-1 tested with Western blot in the outer medullar of the kidneys in the model group decreased.
Conclusions:
1. The animal model of smoking-dosage cadmium was built, contents of renal cadmium in the model group at various time had significant difference compared with the control group, and cadmium was negatively related with calcium and copper.
2. the coefficients of organs in model rats were lower than those of the control group with significant difference at the 20th week , the serum BUN and Crea had difference at the 36th week compared with the control group; the urine Crea had difference at the 36th, 44th and 52nd week compared with the control group.
3. It was observed under the optic microscope that the tissue structure of renal tubule was impaired in the early phase of the model group, and that could be the pathological basis of renal cancer.In the expression of PCNA in the mesangial cells and tubule cells, indicating DNA was impaired in the early stage and repaired gradually, ultimately resulting in hyperplasia.
4. MTs showed significant difference between the two groups, MTs expression was strongest in the treatment group at the 20th week, and attenuated with the model time prolonging, and it was still higher than the control group at the 52nd week. That indicated that the MT induced by cadmium lasted for some time.
5. Protein expression of AQP-1, AQP-2 and AQP-3 in the kidneys of the smoking-dosage cadmium group decreased, effect of the reabsorption of the water.
In conclusion, smoking-dosage cadmium exerted some effect on renal morphology and function, and its damage to the kidneys lasted for a long time. Thus, the research provides scientific theory and experiment foundation in advising people to give up smoking or to avoid Cadmium intake in the smoking population.
方法:建立吸烟相关剂量镉的动物模型,设立模型组和对照组,于20W、28W、36W、44W和52W处死大鼠;应用ICP-MS检测肾脏组织镉、铜、钙、锌元素含量;应用罗氏COBAS Integra 400 Plus全自动生化分析仪检测血清和尿液中BUN、Crea含量及血清钾、钠、钙和氯离子含量;组织切片光镜观察形态学变化;免疫组织化学方法检测MTs及PCNA、AQP-1、AQP-2、AQP-3表达;应用水通透性测定方法检测镉离子对AQP-1的水通透系数;Western blot检测肾脏外髓组织AQP-1蛋白表达。
结果:在吸烟相关剂量镉动物模型组,与对照组比较,各时间点肾脏镉元素含量均有显著差异(P<0.01),并且镉与钙及铜元素之间呈负相关(分别为rs=?0.9,rs=?1.0);与对照组比较,36W时血清BUN、Crea差异明显(P < 0. 05);与对照组比较,尿液肌酐在36W、44W及52W差异显著(P < 0. 05);肾脏组织学观察显示,早期模型组肾脏小管组织结构有一定的破坏,后期则出现上皮不典型增生;吸烟相关剂量镉诱发的MTs定位在肾脏皮质小球、外髓及内髓近曲小管,在细胞胞浆中表达;MTs免疫组化结果表明,模型组20W时表达最强,随模型时间延长逐渐减弱,到52W时仍高于对照组,各时间点模型组与相应对照组比较有显著差异(P<0.01);PCNA在肾小球系膜细胞和小管细胞的表达阳性细胞数呈现先下降随后上升并增多的变化,显示吸烟相关剂量镉干扰了DNA的正常合成;吸烟相关剂量镉模型组肾脏AQP-1、AQP-2及AQP-3蛋白表达降低,随模型时间延长有一定恢复;与对照组比较,直小血管的AQP-1表达差异显著(P<0.01),随模型时间延长逐渐减弱,52W时降至最低;镉对红细胞水通透性没有“门控”作用;Western blot检测模型组肾脏外髓组织AQP-1蛋白表达量下降。
结论:吸烟相关剂量镉可在肾脏持续性累积,造成肾脏功能及形态的损伤,并出现上皮不典型增生,有可能成为肾癌发生的病变基础,其机制可能与镉影响DNA的正常合成有关。该研究提示要重视吸烟相关剂量镉对肾脏的损伤,尤其是不能忽视其远期效应,为说服人们戒烟以减少通过烟草摄入镉提供了理论和实验依据。
The incidence of renal cancer rates second in the urinary system neoplasm, and accounts for 80%-90% of renal malignancies in adults. Renal cancer originates from the epithelia of proximal tubules. Smoking is the main cause of renal cancer, and at least 39% of cases in male are related with it. Cadmium is the major deleterious component in the cigarette and meantime the major pollutant in the environment. The kidneys are the organs impaired most by chronic low-dosage cadmium exposure. Long-term and low-dosage cadmium exposure may impair the epithelia of proximal tubules, resulting in the decreased reabsorption function of proximal tubules. This experiment explored the effect of smoking-dosage cadmium on renal morphology and function.
Objective: to observe the influence of smoking-dosage cadmium on renal morphology and function in rats, and explore the effect of smoking-dosage cadmium on the kidneys and the possible mechanism.
Method: built the animal model of smoking-dosage cadmium; tested the content of cadmium, copper, calcium and zinc in the kidneys with ICP-MS; tested the BUN and Crea content in the serum and urine, as well as the content of potassium, sodium, calcium and chlorine ions in the serum with ROCHE COBAS 400 full-automatic biochemistry analyzer; sliced the tissue and observed the morphologic change under the optic microscope; examined the expression of MT, PCNA, AQP-1, AQP-2 and AQP-3 with immunohistochemical method; measured the water permeability coefficient of cadmium ion to AQP-1 with the water permeability test method; and inspected the protein expression of AQP-1 in the medullary tissue of the kidneys with Western blot.
Results: in the animal model of smoking-dosage cadmium, the coefficients of organs in rats were lower than those of control group(P<0.05)at the 20th week. Compared with the control group, contents of renal cadmium in the model group at various time had significant difference, and cadmium was negatively related with calcium and copper; the serum BUN and Crea had difference at the 36th week compared with the control group; the urine Crea had difference at the 36th, 44th and 52nd week compared with the control group. It was observed under the optic microscope that the tissue structure of renal tubule was impaired in the early phase of the model group, and atypical hyperplasia was observed in the later phase. MTs induced by smoking-dosage cadmium were located in the glomerulus, the outer medullar and proximal tubules in the inner medullar, and expressed in the cell plasma. MTs showed significant difference between the two groups, MTs expression was strongest in the treatment group at the 20th week, and attenuated with the model time prolonging, and it was still higher than the control group at the 52nd week. In the expression of PCNA in the mesangial cells and tubule cells, the positive cell number showed the trend of decreasing first and then increasing, indicating that the smoking-dosage cadmium interfered with the normal synthesis of DNA. Protein expression of AQP-1, AQP-2 and AQP-3 in the kidneys of the smoking-dosage cadmium group decreased, but restored to some extent with the model time prolonging. Protein expression of AQP-1 in the vasa recta had significant difference, attenuated with the model time prolonging and dropped to the lowest at the 52nd week compared with the control group. Cadmium didn’t exert“door control”effect to the water permeability of red cells. Protein expression of AQP-1 tested with Western blot in the outer medullar of the kidneys in the model group decreased.
Conclusions:
1. The animal model of smoking-dosage cadmium was built, contents of renal cadmium in the model group at various time had significant difference compared with the control group, and cadmium was negatively related with calcium and copper.
2. the coefficients of organs in model rats were lower than those of the control group with significant difference at the 20th week , the serum BUN and Crea had difference at the 36th week compared with the control group; the urine Crea had difference at the 36th, 44th and 52nd week compared with the control group.
3. It was observed under the optic microscope that the tissue structure of renal tubule was impaired in the early phase of the model group, and that could be the pathological basis of renal cancer.In the expression of PCNA in the mesangial cells and tubule cells, indicating DNA was impaired in the early stage and repaired gradually, ultimately resulting in hyperplasia.
4. MTs showed significant difference between the two groups, MTs expression was strongest in the treatment group at the 20th week, and attenuated with the model time prolonging, and it was still higher than the control group at the 52nd week. That indicated that the MT induced by cadmium lasted for some time.
5. Protein expression of AQP-1, AQP-2 and AQP-3 in the kidneys of the smoking-dosage cadmium group decreased, effect of the reabsorption of the water.
In conclusion, smoking-dosage cadmium exerted some effect on renal morphology and function, and its damage to the kidneys lasted for a long time. Thus, the research provides scientific theory and experiment foundation in advising people to give up smoking or to avoid Cadmium intake in the smoking population.
引文
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