| The impact of atmospheric fine particulate matter(PM)on public health has become the focus of attention all over the world,especially in developing countries.Initially,the effects of fine particles on respiratory and cardiovascular diseases were emphasized.However,an increasing number of epidemiological studies have shown that every organ system in the body may be involved,and the kidney is no exception.It has been found that PM with aerodynamic diameter less than 10?m(such as PM2.5)has a greater impact on human health.According to epidemiology studies,the prevalence rate of chronic kidney disease(CKD)shows a significant increasing trend,the global prevalence rate is about 10%,the prevalence rate of adult CKD patients in China is 10.8%,and the prevalence rate in Zhengzhou urban area is as high as 13.57%.Its prevention and treatment has become an important public health problem in the world.A great deal of evidence shows that the existence of cardiovascular disease,hypertension and diabetes can predict the rapid progression of CKD to end-stage kidney disease(ESRD),and more and more evidence showed that air pollution may also be a new risk factor for CKD.In addition,studies have shown that long-term exposure to PM2.5 is also associated with the risk of podocytosis such as membranous nephropathy(MN).As an important organ for hemofiltration and excretion of toxins,the kidney is easily affected by air pollutants in the blood,so the effect of PM2.5 exposure on kidney disease can not be ignored.The content of polycyclic aromatic hydrocarbons(PAHs)in atmospheric PM2.5 in China is generally higher than that in foreign countries.It has been reported that benzo[b]fluoranthene(BbF)has the highest content of PAHs in the organic components of atmospheric PM2.5 in Zhengzhou.Our team's previous study found that the content of BbF,the principal component of PM2.5,was significantly increased in patients with IMN.It is speculated that BbF is involved in the occurrence and development of IMN.Autophagy is an intracellular degradation system that maintains homeostasis by transferring cytoplasmic components to lysosomes.Autophagy is mediated by a unique organelle called autophagosome.Dysfunction of autophagy formation in podocytes can lead to podocyte dysfunction and glomerular disease.It is reported that autophagy may play an important role in the cytotoxicity induced by benzo[a]pyrene(a common polycyclic aromatic hydrocarbon).However,as the most toxic component of PAHs,the autophagy damage effect of BbF,on mouse glomerular podocytes is not clear.Cell pyroptosis is a special way of cell inflammatory death newly discovered in recent years.Cell pyroptosis is affected by many factors,autophagy is one of the important mechanisms.Moreover,the inhibition of autophagy in renal disease is often related to the occurrence of cell pyroptosis.Based on this,we hypothesized that BbF enters the body from the lungs and enters the kidney through blood circulation,which inhibits podocyte autophagy and induces NLRP3 inflammatory body activation and podocyte injury.In this study,cellular and molecular biological techniques were used to observe the effects of different concentrations,doses and exposure time of BbF on renal podocyte autophagy and NLRP3 inflammatory body activation,and to explore the molecular mechanism of podocyte autophagy induced podocyte injury in BbF,so as to provide new ideas for its prevention and treatment.MethodsIn this study,cultured conditionally immortalized mouse podocytes(MPC)was selected as research objects.1.Cell Counting Kit-8(CCK-8)was used to determine the effects of BbF exposure on the cell viability of MPC under different concentrations(0 ?g/ml,6.25?g/ml,12.5 ?g/ml,25 ?g/ml,50 ?g/ml,100 ?g/ml)and different time(0h,6h,12h,48h,72h,96h).2.The podocyte injury in vitro after exposure to BbF in different concentrations(0 ?g/ml,6.25 ?g/ml,12.5 ?g/ml,25 ?g/ml)and different time(0h,24 h,48 h,72 h):the morphological changes of podocytes were observed under light microscope,the changes of cytoskeletal protein F-actin and Synaptopodin were observed by immunofluorescence,and the expression of the slit diaphram proteins such as Nephrin,Podocin and the intermediate filaments such as desmin(podocyte injury marker)were observed by western blotting analysis.3.The autophagy of podocytes after exposure to BbF in different concentrations(0 ?g/ml,6.25 ?g/ml,12.5 ?g/ml,25 ?g/ml)and different time(0h,24 h,48 h,72 h):the autophagosomes of podocytes were observed under transmission electron microscope,and the expression of autophagy-associated proteins were observed by western blotting and immunofluorescence.4.Effects of Rapamycin,an inducer of autophagy,and Chloroquine,CQ,an autophagy inhibitor,on podocyte injury and autophagy:podocytes were divided into control group,BbF group,CQ group,BbF+CQ group,rapamycin group and BbF+rapamycin group.The changes of cytoskeletal protein synaptopodin were observed by immunofluorescence,and the expression of Nephrin and autophagy-associated proteins LC3B and Beclinl were observed by western blotting.5.The activation of NLRP3 bodies in podocytes after exposure to BbF in different concentrations(0 ?g/ml,6.25 ?g/ml,12.5 ?g/ml,25 ?g/ml):western blotting was used to observe the expression of related indexes(NLRP3 and caspase-1)after activation of NLRP3 inflammatory bodies in different groups of podocytes to explore the role of NLRP3 inflammatory bodies in podocyte injury.Results1.CCK-8 results showed that podocytes viability reduced dose-and time-dependently after exposure to BbF(P<0.05).2.The results of light microscope showed that the morphology of podocytes changed with the increase of concentration and time.The results of cellular immunofluorescence showed that the expression intensity of cytoskeletal proteins in podocytes decreased with the increase of exposure concentration and time of BbF.The results of western blotting analysis showed that with the increase of exposure concentration and time of BbF,the podocyte injury was more serious,the expression of Synaptopodin,Nephrin and Podocin gradually decreased,while the expression of Desmin increased,indicating that the renal injury was aggravated in a dose-and time-dependent manner(P<0.05).3.Western blotting analysis showed that with the increase of exposure concentration and time of BbF,the expression of autophagy-associated proteins Beclinl and LC3B decreased,and positively correlated with reduced expression of Nephrin.The results of transmission electron microscope showed that the number of autophagosomes in podocytes decreased,indicating that the level of autophagy decreased with the increase of concentration(P<0.05).4.The results of western blotting analysis and immunofluorescence showed that the autophagy of podocytes induced by CQ was alleviated and the podocyte injury was aggravated.Under the intervention of rapamycin,autophagy was aggravated and podocyte injury was alleviated by BbF exposure.5.The results of western blotting analysis showed that with the increase of BbF concentration,the related indexes of NLRP3 inflammasomes activation(NLRP3 and caspase-1),indicating that podocyte injury caused by BbF may be related to the activation of NLRP3 inflammasomes.Conclusions1.Benzo[b]fluoranthene,an organic component of atmospheric fine particles,caused podocyte injury in dose-and time-dependent manner.2.Autophagy inhibition and NLRP3 inflammasomes activation are the possible mechanisms of kidney disease induced by benzo[b]fluoranthene induced podocyte injury,which need to be further explored. |
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