Cadmium Induced Apoptosis Of Human Renal Tubular Epithelial Cells Via ROS-mediated Oxidative Damage

In order to study the toxic effects of Cadmium?Cd?exposure on human renal tubular epithelial cells?HK-2?,the effects of Cd Cl₂ on oxidative damage and apoptosis of HK-2 cells was tested by MTT assay,light microscope observation,flow cytometry,fluorescence staining,comet assay and Western blot.HK-2 cells were co-treated with Cd Cl₂ and NAC to explore the role of ROS in oxidative damage and apoptosis of HK-2 cells caused by Cd Cl₂.As well as a mice Cd poisoning model was established,detecting the activities of several important antioxidant enzymes and the content of lipid peroxides in mice kidney tissues,observing the changes of the microstructure of kidney tissues by HE staining,and counting the kidney coefficient of mice.The purpose of the study was to explore the toxic mechanism of Cd exposure on kidney injury and provide theoretical basis for prevention of heavy metal Cd poisoning and clinical treatment.The experimental results are as follows:The first,in vitro experiments discovered that compared with the control group,Cd Cl₂ treatment significantly reduced the viability of HK-2cells,showing an obvious time-dose-effect relationship;The morphology of HK-2 cells was observed by optical microscope.It was found that HK-2 cells gradually shrunk,rounded and shed after Cd Cl₂ treatment;Fluorescence microscopy and flow cytometry showed that after Cd Cl₂ treatment,ROS content in HK-2 cells increased significantly and mitochondrial membrane potential decreased;Comet assay and Western blot results showed that Cd Cl₂ treatment could caused DNA fragmentation of HK-2 cells and significantly increase the expression of ?-H2 AX protein,indicating that Cd Cl₂ caused DNA damage of HK-2 cells and was related to ROS generation;Hoechst staining also showed that HK-2 cells appeared chromatin condensation,nuclear condensation and fragmentation;Western blot results showed that caspase-3 was activated after Cd Cl₂ treatment,which indicated that Cd treatment could cause mitochondrial damage and apoptosis in HK-2 cells.Compared with the Cd Cl₂ treatment group,the co-treatment of NAC and Cd Cl₂ could reduce cell morphological changes;Inhibition of intracellular ROS production;Moreover,the degree of DNA damage and cell apoptosis were reduced,indicating that the toxic damage of Cd Cl₂ to HK-2 cells was related to ROS.The second,animal experiments showed that compared with the control group,the activities of GSH-Px and CAT in kidney tissue of mice treated with Cd Cl₂ decreased significantly at 19 d;SOD activity increased first and then decreased with the increase of concentration under the same treatment time.MDA content showed an increasing trend;HE staining results showed that renal tubular epithelial cells showed granular degeneration,partial cell exfoliation or necrosis.Conclusions:Firstly,Cd has caused ROS accumulation in large quantities,thus causing a series of oxidative stress reactions such as decrease of mitochondrial membrane potential and DNA damage in HK-2 cells,and eventually leading to apoptosis of cells.Preliminary results have showed that Cd could caused apoptosis of renal tubular epithelial cells through ROS-mediated oxidative damage.Secondly,Cd exposure has destroyed the activity of antioxidant enzyme system in mice kidney tissue,leading to damage or even necrosis of renal tubular epithelial cells,further proving that oxidative damage could produced toxic effects on renal tubular epithelial cells.