The Effects And Mechanism Of Low Concentration Cadmium On The Function Of Human Renal Mesangial Cells And Renal Podocytes

BackgroundCadmium(Cd)is a common environmental toxins and industry pollutants.It can enter body through contaminated food,drinking water,smoking and occupational exposure.Due to long biological half-life,It can accumulate in different target organs.Therefore,Cd exposure can cause serious damage to human health.it can produce toxic effects to bone,kidney,liver,reproductive and immune organs.The kidney is an important target organs of Cd.It can make the liver produce Metallothionein(MT),and bind to it.Cd-MT is transferred to the kidney and other target organs with the blood.Cd can damage glomeruli and proximal convoluted tubule and distal convoluted tubules by stress response.Cd exists in the blood circulation with free ions or the form of combination with plasma protein.Cd is filtered by glomeruli and can directly damage it causing proteinuria.Cd accumulation can also result in renal tubular resorption dysfunction.Human glomerular cells are mainly composed of human renal glomerular endothelial cells(HRGECs),human renal mesangial cells(HRMCs)and human renal podocytes(HRPs).Our previous study found that low concentration Cd had no significant effect on function in HRGECs,but the effect of low concentration cadmium on HRMCs and HRPs is not clear,and need further research.C-Jun amino terminal kinase(JNK)family is one of the number of mitogen activated protein kinases(MAPKs),and can be activated by a variety of factors such as cytokines,growth factors,stress reaction.JNK participates in a series of biological responses including cell proliferation and differentiation,cell morphology and cell skeleton construction.Activated JNK transfers to the nucleus and activates c-Jun,leading to the formation of a heterodimer of c-Fos and c-Jun which are the most common members of the activator protein-1(AP-1)family.These transcription factors can combine many gene promoter regions activating the AP-1 site and increase the transcriptional activity of specific genes.Many studies have shown that a variety of stress signal can activate the JNK signaling pathway and regulate the expression levels of many stress-response genes.It is unclear whether Cd affects the function of HRMCs and HRPs through activating the JNK signaling pathway.Therefore we have set up a cell model of stimulating HRMCs and HRPs in vitro with Cd,and observe the effect of low concentration Cd on HRMCs and HRPs,and to explore JNK signaling pathways in the role of the effect of low concentration Cd on HRMCs and HRPs.Our study will provide a new thought for the treatment of Cd related renal diseases.Objective1.To explore the effects of low concentration Cd on HRMCs and the role of JNK pathway in the changes induced by Cd.2.To explore the effects of low concentration Cd on HRPs and the role of JNK pathway in the changes induced by Cd.Methods1.The effects and mechanism of low concentration Cd on the function of HRMCs1.1 The effects of low concentration Cd on proliferation and viability of HRMCsAfter treated with Cd for 24 h.HRMC proliferation was evaluated by an MTT assay,cell counting,and the expression levels of(proliferating cell nuclear antigen,PCNA).Cell viability was assessed by trypan blue exclusion assay.1.2 The effects of low concentration Cd exposure on the mesangial cell markersAfter exposure to Cd for 24 h,the characteristics of HRMCs were examined by immunofloursence staining for a-SMA and PDGFR-?.1.3 The effects of low concentration Cd exposure on the F-actin cytoskeleton of HRMCsWe examined F-actin with Phalloidin staining in HRMCs monolayer treated with PBS(control);Cd for 24 h;SP600125 for 24 h;Cd and SP600125 for 24 h.1.4 The effect of low concentration Cd on JNK signaling pathway of HRMCsAfter exposure to Cd for 0 h,1 h,2 h,6 h,12 h,24 h,The protein of HRMCs was extracted and observed the expression levels of Total JNK;p-JNK;c-Jun and c-Fos.1.5 The effect of JNK inhibitor on the proliferation of HRMCs treated with CdSP600125 was applied to cells 1 h before the treatment of Cd for 24 h.HRMC proliferation was evaluated by an MTT assay.The expression levels of PCNA were examined by Western blotting.2.The effects and mechanism of low concentration Cd on the function of HRPs2.1 The effects of low dose Cd exposure on the F-actin cytoskeleton of HRPsWe examined F-actin with Phalloidin staining in HRPs monolayer treated with PBS(control);Cd for 24 h;SP600125 for 24 h;Cd and SP600125 for 24 h.2.2 Effects of Cd on proliferation,viability and the expression of cell type-specific markers of HRPsAfter exposure to Cd for 24 h,HRP proliferation was evaluated by an MTT assay.Cell viability was assessed by trypan blue exclusion assay.The characteristics of HRPs were examined by immunofloursence staining for CD2AP and Synaptopodin.2.3 The effect of low concentration Cd on JNK signaling pathway of HRPsAfter exposure to Cd for 0 h,1 h,2 h,6 h,12 h,24 h,The protein of HRPs was extracted and observed the expression levels of Total JNK;p-JNK;c-Jun and c-Fos.2.4 The effect of JNK inhibitor on the proliferation and viability of HRPs treated with CdSP600125 was applied to cells 1 h before the treatment of Cd for 24 h.HRMC proliferation was evaluated by an MTT assay.Cell viability was assessed by trypan blue exclusion assay.Results1.The effects and mechanism of low concentration Cd on the function of HRMCs1.1 Low concentration Cd inhibits the proliferation of HRMCThe effect of low concentration Cd on the proliferation of HRMC was examined by MTT assay,cell counting and the expression levels of PCNA.We found that Cd decreased the MTT OD reading(p<0.01)and the cell counting of HRMCs(p<0.01).PCNA,a marker of proliferation,was decreased in HRMCs exposed to Cd for 24 h as shown by immunoblots densitometry analysis(1 vs.0.59 ± 0.02,p<0.01).However,trypan blue exclusion assay showed that Cd does not affect viability of the HRMCs(p=0.219).The results indicates that Cd decreased the proliferation of HRMCs.1.2 The low concentration Cd has no significant effect on the expression of a-SMA and PDGFR-p of HRMCsIn this study,the characteristics of HRMCs were examined by immunofloursence staining for a-SMA and PDGFR-?.We found that the expression of a-SMA and PDGFR-? in HRMCs were not altered after 24 h exposure to Cd.Therefore,low dose Cd does not induce transdifferentiation of HRMCs.1.3 The low concentration Cd had no significant effect on the expression and distribution of F-actin in HRMCsWe examined the F-actin cytoskeleton of mesangial cells by exposing the cells to its interacting molecule phalloidin that was conjugated to a fluorochrome.We found that F-actin arrangement in HRMCs was not disrupted compared to controls,following 24 h exposure to Cd.In addition,a combined treatment with SP600125 and Cd did not significantly change the F-actin arrangement in HRMCs compared to the cells with SP600125 alone.1.4 Low concentration Cd activates JNK pathway in HRMCsIn this study,we examined whether low dose Cd exposure activates the JNK pathway in HRMCs by Western blotting.The phosphorylated JNK was significantly increased in HRMCs treated with Cd,while the total levels of JNK protein remained unchanged.Moreover,protein levels of c-Fos and c-Jun,the downstream effectors of the JNK pathway,were significantly increased.Therefore,low dose Cd activates JNK pathway in HRMCs and increases the expression of phosphorylated JNK,c-Fos and c-Jun.1.5 Low concentration Cd inhibits proliferation of HRMC via activation of JNK pathwayPretreatment with SP600125 for 1 h inhibited phosphorylation of JNK in HRMCs exposed to Cd.We also found that the MTT OD reading,cell counting and PCNA level of HRMCs treated with a combination of SP600125 and Cd was similar to that of treatment with SP600125 alone(p = 0.655,p = 0.657,p = 0.938,respectively).Thus,the JNK pathway mediates the Cd induced decrease in HRMC proliferation.2.The effects and mechanism of low concentration Cd on the function of HRPs2.1 Low concentration Cd had no significant effect on the expression and distribution of F-actin in HRPsWe examined the F-actin cytoskeleton of podocytes by exposing the cells to its interacting molecule phalloidin that was conjugated to a fluorochrome.We found that F-actin arrangement in HRPs was not disrupted compared to controls,following 24 h exposure to Cd.2.2 Low concentration Cd exposure does not affect proliferation,viability and the expression of cell type-specific HRP markersThe MTT assay indicated that Cd did not significantly inhibit HRP proliferation after 24 h.The results of the trypan blue exclusion assay also demonstrated that HRP viability remained unchanged following Cd treatment.HRP characteristics were examined by immunofluorescence staining for CD2AP and synaptopodin.CD2AP and synaptopodin expression was unchanged in HRPs following 24 h exposure to Cd.These results suggested that low dose Cd exposure may not alter the proliferation,viability and phenotype of HRPs.2.3 Low concentration Cd activates JNK pathway in HRPsWestern blotting indicates that the phosphorylated JNK was significantly increased in HRMCs treated with Cd,while the total levels of JNK protein remained unchanged.Moreover,protein levels of c-Fos and c-Jun were significantly increased.2.4 The JNK inhibitor had no significant effect on the function of the HRPs treated with CdHRP proliferation and viability were similar in the group treated with a combination of SP600125 and Cd compared with in the group treated with SP600125 alone.In addition,SP600125,or a combined exposure to SP600125 and Cd,did not significantly alter F-actin arrangement in podocytes.Conclusion1.Cd treatment induces a decrease in proliferation.In addition,Cd does not change the expression of a-SMA and PDGFR-? or the alignment of the F-actin cytoskeleton of HRMCs.Cd activates JNK pathway in HRMCs,and the decrease in HRMC proliferation was reversed by pretreatment with SP600125,an inhibitor of the JNK pathway.2.The expression levels of CD2-associated protein and Synaptopodin are unchanged or the alignment of the F-actin cytoskeleton of HRPs following Cd treatment.Although Cd activates JNK pathway in HRPs,proliferation and viability in HRPs were not significantly affected by Cd treatment.