Molecular Mechanisms Of Cadmium And Dihydroartemisinin On The Permeability Of Glomerular Endothelial Cells

Background:With the development of industry,environmental toxins and food safety problems have become significant health risks to humans.Cadmium(Cd)is a common heavy metal pollution,and was identified as one of the carcinogens by the international agency for research on cancer(IARC)in the 1990s.With a long biological half-life and low excretion rate,it can accumulate in different target organs,such as kidney,liver,lung,cardiovascular system and immune system.Kidney is one of the major targets of Cd toxicity.Acute or chronic Cd exposure can both affect the excretion mechanism of the kidney.After respiratory or oral exposure,Cd enters the human body and is absorbed into the bloodstream.Before it is taken up by kidney,Cd circulates in the blood as a bound with the metallothionein(MT)which produced by liver.Through systemic circulation,plasma Cd interacts with glomerular endothelial cells,the inner layer of blood vessels,and may be have a direct effects on the endothelium.Cd can not only damage endothelial cells directly,but also destroy the cellular connection structure between endothelial cells and increase its permeability by destructing endothelial cells junctions,and thus resulting in proteinuria.The adhesion junction plays a maj or role in regulating the paracellular permeability.Vascular endothelial(VE)-cadherin is a major component of endothelial adhesion junction,and VE-cadherin/?-catenin complexes mediate the distribution of adhesion junction by interacting with p38 mitogen activated protein kinase(MAPK).Thus,we cultured human renal glomerular endothelial cells(HRGECs)in the presence of serum with treatment of a short term(1 h)and low-dose(1 ?M)(ST/LD)of Cd,and explored the effects of cadmium on the permeability of HRGECs at specific doses and times.Objective:(1)To explore the effects of ST/LD cadmium on the permeability of human renalglomerular endothelial cells.(2)To explore the role of p38 MAPK signaling in regulating the effects of ST/LDcadmium on the permeability of human renal glomerular endothelial cells.Methods:(1)Stimulation of cellsCdCl2 with a final concentration of 0,0.1,1 and 10 ?M was used to stimulate the HRGECs.The ST/LD treatment is defined as the exposure of 1 ?M CdCl2 for 1 h.To confirm the impact of the p38 MAPK signaling pathway on HRGECs,inhibitors of the MAPK pathway PD98059,SP600125 and SB203850 were added 1 hour earlier than CdCl2 at concentration of 10 ?M.(2)Cell Proliferation and Viability AssayMTT and Trypan blue exclusion assay were used to detect the proliferation ability and cell viability of HRGECs exposed to CdCl2at concentration of 0,0.1,1 and 10?M,respectively.(3)Annexin V-Fluorescein Isothiocyanate/Propidium Iodide AnalysesFlow cytometry analyses were used to detect the apoptosis rate of HRGECs treated with ST/LD cadmium.(4)Transwell AssayFITC-dextran transwell assay was used to detect the permeability of HRGECs exposed to CdCl2 at concentration of 0,0.1,1 and 10 ?M,respectively.(5)Electric cell-substrate impedance sensing,ECIsCells were seeded into ECIs plates and divided into control group(PBS),CdCl2 group(ST/LD),CdCl2+PD98059 group,CdCl2+SP600125 group,CdCl2+SB203850 group.The real-time transendothelial electrical resistance measurement of HRGECs was detected to reflect their permeability changes.(6)Quantitative Real-Time Polymerase Chain ReactionCells were divided into control group(PBS),CdCl2 group(ST/LD).The RNA of each group was extracted and the CDH5?CTNNB1 gene expression was detected by reverse transcription and RT-qPCR.(7)Western BlotCells were divided into control group(PBS),CdCl2 group(ST/LD),CdCl2+PD98059 group,CdCl2+SP600125 group,CdCl2+SB203850 group.The protein of each group was extracted and observed the expression levels of MAPK signaling pathway.(8)ImmunofluorescenceCells were divided into control group(PBS),CdCl2 group(ST/LD),CdCl2+SB203850 group.Each group was examined by immunofluorescence staining for the expression and distribution of VE-cadherin and ?-catenin protein.Results:(1)MTT and trypan blue assay showed that when concentration of cadmium was lower than 10 ?M,CdCl2 had no obvious effect on the proliferation ability and viability.Flow cytometry analyses exhibited that ST/LD cadmium exposure did not significantly affect the apoptosis rate to HRGECs.(2)Immunofluorescence staining revealed that ST/LD cadmium exposure could dislocate VE-cadherin and its binding protein ?-catenin from the cell membrane of HRGECs.While inhibit p38 MAPK signaling pathway could suppress ST/LD Cd-induced the redistribution of VE-cadherin and ?-catenin.(3)Transwell Assay showed that the final concentration of 1 ?M and10 ?M CdCl2 could both increase the permeability of HRGECs.(4)Western Blot experiments showed that ST/LD cadmium exposure could activate MAPK signaling pathway.While the inhibitors of PD98059,SP600125,SB203850 could successfully inhibite ST/LD Cd-induced activation of MAPK signaling pathway.(5)ECIs results showed that SB203850 could effectively reverse Cd exposure-induced increase of endothelial cells permeability.Conclusion:ST/LD cadmium exposure increases permeability of HRGECs through the dislocation of adhesion junction proteins VE-cadherin and ?-catenin without cytotoxic effects.Blocking the p38 MAPK signaling pathway can effectively reduce Cd exposure-induced vascular toxicity and provide a new strategy for clinical treatment.Background:VE-cadherin is the key molecule in endothelial cells(EC)adherens junction,and also plays a critical role in mediating EC adherens junctions,endothelial permeability and intracellular signal transduction pathways.In recent years,VE-cadherin has received extensive attention for the treatment of kidney disease.More and more evidence indicates that VE-cadherin is widely involved in the development of various kidney diseases,especially in regulating the permeability of glomerular microvascular endothelium and the reduction of proteinuria.Artemisia annua is a unique herbaceous plant and has a long history of medicine use in China at least 2000 years.Artemisinin is a sesquiterperne lactone endoperoxide extracted from the Artemisia annua.It is widely used as an anti-malarial with few adverse side effects.Dihydroartemisinin(DHA)is a semi-synthetic derivative of Artemisinin,and has the advantages of fast absorption,wide distribution,low toxicity and quick excretion in the body.It has anti-malarial,anti-tumor,anti-fungal,anti-angiogenic,anti-inflaminatoiy and other pharmacological effects.Researches showed that DHA could inhibit the proliferation,migration and tube formation of human umbilical vein endothelial cells(HUVECs).However,the effect of DHA on endothelial cells permeability is not clear.In the study,we further explored the effects of DHA on endothelia cells permeability.We found that DHA specifically increases VE-cadherin expression,reduces the hyperpermeability of glomerular endothelial cells,and thus prevents the proteinuria to some extent.This study expanded our understanding regarding the molecular mechanism underlying DHAs anti-hyperpermeability effects and supported the potential use of DHA as an agent for the treatment of chronic kidney disease.Objective:(1)To confirm the effects of DHA on the human renal glomerular endothelial cells permeability.(2)To explore the molecular mechanisms of DHA anti-hyperpermeability effects on the human renal glomerular endothelial cells.Methods:(1)Western BlotCells were divided into control group(PBS),DHA group(25 p,M).The protein of each group was extracted after 24 hs and observed the expression levels of VE-cadherin,Snail?Slug,Smad2/3 and phosphorylated Smad2/3.(2)Quantitative Real-Time Polymerase Chain ReactionCells were divided into control group(PBS),DHA group(25 p,M).The RNA of each group was extracted after 24 hs and the VE-cadherin,Snail,Slug gene expression was detected by reverse transcription and RT-qPCR.(3)ImmunofluorescenceCells were divided into control group(PBS),DHA group(25 p.M).Each group was examined by immunofluorescence staining for the expression and distribution of VE-cadherin protein.(4)Chromatin Immunoprecipitation(ChIP)To obtain the optimum length DNA fragment,ultrasound was use to lyse HRGECs by DHA treatment.After adding Snail,Slug antibody to obtain target DNA and purification respectively,VE-cadherin promoter was used for PCR amplification to detect the binding of Snail,Slug and VE-cadherin promoter.Results:(1)Western blot and RT-qPCR experiments showed that DHA specifically up-regulated the expression of VE-cadherin protein and mRNA in HRGECs.(2)Western Blot analysis showed that the expression of TGF-(3 RI and p-Smad2/3protein were significantly decreased,while the expression of total protein remained unchanged.It confirmed that DHA could inhibit the activation of TGF-? RI signaling pathway.(3)Western Blot and RT-qPCR experiments showed that DHA specifically up-regulated the expression of Snail,Slug protein and mRNA in HRGECs.(4)Chip experiments finally confirmed that DHA could specifically reduce the binding of Snail,Slug with VE-cadherin promoter,increase the activity of the VE-cadherin promoter,and thus up-regulate its expression.Conclusion: DHA-induced increase in VE-cadherin expression arises through down-regulation of basal levels of transforming growth factor receptor I(TGF-? RI)-Smad2/3 signaling,lower expression of Snail and Slug and ultimately a relief of transcription repression of the VE-cadherin promoter.Thus,DHA regulates glomerular permeability by elevation of VE-cadherin expression.Background:Cadmium is a heavy metal pollutant which can enter the human body through the food chain and pose significant health risks to humans.Cadmium can induce distinct pathological changes in a variety of organs,such as kidney damage,cardiovascular damage,bone damage,immune system damage and tumor.Kidney is the main target organ of cadmium chronic toxicity.Cadmium accumulates in the proximal and part of the distal convoluted tubules,resulting in tubular impairment with a loss of absorptive capacity and development of proteinuria.Cadmium travels in the blood circulation as a bounded with a low-molecular weight metallothionein,which can directly interact with the glomerular vascular endothelial cells and affect the function of glomerular filtration barrier.Studies show that cadmium induces cell death in a variety of cell types,but the effect of cadmium on glomerular endothelial cells is still unclear.NF-?? is a nuclear transcription factor and widely exists in eukaryotic cells.At rest,it resides in the cytosol,binds to the inhibitory protein inhibitor of IkBol When stimulated,IkB kinase will be phosphorylated and proteolysis.Following the removal of IkB,NF-?? enters the nucleus to induce the expression of targets genes for a variety of pathophysiological processes.It also plays an important role in cell proliferation,differentiation,apoptosis and autophagy.In this study,we evaluated the effect of low dose cadmium on apoptosis of human renal glomerular endothelial cells(HRGECs).We selected the NF-?? inhibitor PDTC to explore the effect of blocking NF-?? signaling on the survival of HRGECs stimulated by cadmium and its underlying mechanisms.Objective:(1)To confirm the effects of cadmium on the apoptosis of human renal glomerular endothelial cells.(2)To confirm the role of NF-?? signaling in regulating cadmium induced apoptosis of human renal glomerular endothelial cells.Methods:(1)Stimulation of cellsHRGECs were tested 24 h after stimulation with a final concentration of 4 \iM CdCb.To confirm the impact of NF-??/JNK signaling on HRGECs,inhibitors of PDTC/ SP600125 were added 1 h earlier than CdCl2 at concentration of 10(2)Cell Viability AssayCells were divided into control group,CdCh group,CdCU+PDTC group.Trypan blue exclusion assay was used to detect the viability of each group.(3)Annexin V-FIuorescein Isothiocyanate/Propidium Iodide AnalysesCells were divided into control group,CdCh group,CdCh+PDTC group,CdCl2+PDTC+SP600125 group.Flow cytometry analyses were to detect the apoptosis rate of each group.(4)Western BlotThe proteins extracted from HRGECs which exposed to CdCh at a concentration of 4 jjM for different time points,and detected the expression of IkB protein;Other proteins were extracted by control group and CdCb+PDTC group,and observed the expression levels of phosphor-JNK or JNK protein.(5)ImmunofluorescenceCells were divided into control group,CdCb group.Each group was examined by immunofluorescence staining for the expression levels of NF-?? p65 protein.(6)Statistical AnalysisSame as the first part.Results:(1)Western Blot analysis revealed that the protein level of iKBa in the cytoplasm of HRGECs was significantly decreased by cadmium exposure.Immunofluorescence staining demonstrated that cadmium exposure could increase the NF-?? p65 protein translocated into nucleus.(2)Flow cytometry analyses exhibited there were no significant changes in apoptosis and cell activity in HRGECs treated with 4 jiM cadmium or PDTC.However,when cadmium and PDTC were simultaneously treated with HRGECs,the apoptosis rate increased and the cell activity decreased significantly.These findings demonstrated that NF-?? signaling pathway was required for the survival of Cd-stimulated HRGECs.(3)Western Blot analysis showed that cadmium increased phosphorylated(p-)JNK in HRGECs.Densitometry analyses showed that the combination of PDTC and Cd significantly elevated p-JNK compared with exposure to Cd alone.Thus,the JNK pathway was suppressed by Cd-activated NF-?? signaling pathway.(4)Flow cytometry analyses demonstrated that the addition of SP600125 partially reversed the apoptosis of HRGECs.This suggested that NF-?? partially inhibited the Cd-induced apoptosis of HRGECs through the negative regulation of the JNK pathway.Conclusion:Cd activates the NF-?? pathway and inhibits the JNK pathway to maintain the survival of HRGECs exposed to Cd.