Effects Of Cadmium Chloride On Mitochondrial Function And Expression Of PGC-1??NRF-1 In HK-2 Cells

BackgroundCadmium?Cd?and its compounds are common heavy metal pollutants in the current environment,which mainly come from industrial production and agricultural application.Kidney as one of the main accumulation organ of cadmium,proximal tubule of the S1segment and S2 segment is the main target of cadmium toxicity.The mechanism of the effect of cadmium chloride on the injury of human renal tubular epithelial cells?HK-2?has not yet been elucidated.The disorder of mitochondrial structure and function may be an early biomaker of cadmium-induced renal cytotoxic injury.As a nuclear-encoded transcriptional coactivator,PGC-1?can enhance nuclear receptor and related transcriptional activity,plays an important regulatory role in mitochondrial biogenesis and mitochondrial function.However,the effect of PGC-1??NRF-1 on the mitochondrial toxicity of HK-2 cells induced by CdCl₂ is not yet clear.ObjectiveIn this study,to observe the effects of cadmium chloride on mitochondrial function and the expression of PGC-1??NRF-1 protein in HK-2 cells,and explore the possible role of cadmium chloride in the process of mitochondrial oxidative damage of HK-2cells.Methods1.In this study,HK-2 cells were cultured in vitro.When cells growth in logarithmic state,we divided them into control and experimental group.The latter was divided into groups with Cadmium chloride at different concentrations?10,20,30,40,50,60?mol/L?.Cells were collected after continuous exposure for 24h,MTT assay was used to observe the viability of HK-2 cells.2.The mitochondrial ROS was detected by MitoSOX?Red staining,which observed by Confocal microscopy.The changes of mitochondrial ROS fluorescence intensity were analyzed by Fluorescence quantitative analysis.3.Mitochondria of cells were extracted and the activity of respiratory chain complex III was determined by Multi-functional microplate reader.4.The changes of mitochondrial membrane potential was measured by JC-1staining flow cytometry.5.The expressions of PGC-1??NRF-1 in HK-2 cells were detected by Western blot analysis.Results1.Compared with the control group,at the dose of 10?mol/L,HK-2 cells grew nor-mally?P=0.089?;while at the dose of 20⁶0?mol/L,the survival rate of HK-2 cells was obviously decreased from?77.60±0.8185?%to?41.96±1.2220?%?P<0.01?,and showed a significant dose-response relationship.2.Compared with the control group,at the dose of 20?mol/L CdCl₂,the changes of MitoSOX?Red staining were gradually increased in HK-2 cells;Fluorescence quantit-ative analysis showed that compared with control group,at the dose of 20?mol/L,the av-erage density of mitochondrial ROS fluorescence was significant difference?P<0.05?,at the dose of 30⁶0?mol/L,the difference increased obviously?P<0.01?.3.Compared with the control group,the results showed that at the dose of20?mol/L,the activity of respiratory chain complex III was decreased?P<0.05?;especi-ally at the dose of 40⁶0?mol/L,there were obviously decreased?P<0.01?.4.Compared with the control group,the JC-1 monomers positive ratio by the perce-ntage of the treated cells?20⁶0?mol/L?was 1.5100±0.1353,1.5800±0.1670,1.7167±0.1305,2.4100±0.1539,3.4733±0.2702 higher than untreated control?P<0.01?.It is shown that mitochondrial membrane potential decreased gradually.5.Compared with the control group,at the dose of 20?mol/L,the expression of PGC-1?was decreased?P<0.05?,at the dose of 30⁶0?mol/L,the difference wasdecreased obviously?P<0.01?;at the dose of 20⁶0?mol/L,protein expression of NRF-1was also decreased significantly?P<0.01?.Conclusion1.At the dose of 20⁶0?mol/L,cadmium chloride can inhibit the survival of HK-2cells and shows a dose-response relationship;2.Cadmium chloride may inhibit the expression of PGC-1?and the activity of respiratory chain complex III,and by inducing the mitochondrial ROS production and reducing mitochondrial membrane potential,leading to the damage of HK-2 cells.