Mechanism Of Podocyte Autophagy Promoted By Neutrophil Extracellular Traps And The Effect Of Sinomenine

Podocytes,as highly differentiated cells,are important constituents of the glomerular filtration barrier,which prevent the loss of plasma proteins and have numerous critical biological functions.Podocytes are often exposed to various damaging factors,which can potentially induce oxidative stress and DNA damage.Injured podocytes play a key factor in the pathogenesis of glomerular diseases.Neutrophils can be recruited by podocytes to inflammation tissues and taken anti-inflammation effect,but glomerulus will be injured if this process is out of control or excessively promoted.Actived neutrophils also regulate other elements of the immune system.Neutrophil extracellular traps(NETs)are a novel form of killing microorganism through releasing lysozyme and chromatin.But excessive NETs formation or degradation was defected would led to expose various antigens during neutrophil activation,and this process can break self tolerance.Autophagy is a very important intracellular degradation and stability for internal environment,and it aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress.Whereas,excessive high level of autophagic may damage proteins and organelle in cell and finally lead to cell death.Podocytes are cells with high levels of basal autophagy.A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli during autophagy.NOX is one of the major sources of ROS.Podocytes generate ROS via NADPH oxidase and express p47-phox subunit,a major regulator subunit which is required for NOX full activation.Angiotensin II(Ang ?)is the major effect factor in renin-angiotensin system.Podocytes not only have the Ang ? receptor,but are able to secrete Ang ? by autocrine.Sinomenine hydrochloride(SN)is an alkaloid that has effects of immunosuppression and anti-inflmmation.Recently research found that SN has the ability of reducing LPS-induced ROS in neuronglia cells.Based on the above findings,we had observed the influence of Ang ? secretion by podocyte under stimulation of NETs,and further investigated the effect of SN in Ang ?-induced podocytes.Materials and Methods1.Recruitment and isolation of NETs-prone neutrophils by subcutaneously implanted agarose gel.Mouse podocytes and conditionally immortalized human podocytes(AB8/13)were cultured in medium.2.The morphology of neutrophil and NETs formation were investigated by immunofluorescence.The expression of p47-phox subunit in membrane was investigated by immunofluorescence.3.The purity of cells was analyzed by flow cytometer.The ROS produced by podocytes was also analyzed by flow cytometer.4.The concentration of Ang ? secreted by podocytes under stimulation with NETs was detected by ELISA.5.LC3 B protein and p47-phox membrane protein were detected by Western blot.6.The formation of autophagosome was observed by Transmission electron microscope.Results1.A large number of high purity(reach to 95.7%)NETs-prone neutrophils could be elicited by garose gel subcutaneously implanted in mouse.2.NETs promoted Ang ? secreted by podocytes(p=0.027).3.SN reduced NETs formation in neutrophils(p=0.002).4.SN self did not promote LC3 B in podocytes(p>0.05).5.Ang ? significantly promoted LC3 B in podocytes(P<0.05),and promoted autophagosomes formation(P<0.001).SN was able to attenuate LC3 B expression and autophagosomes formation(P<0.05 and P<0.001,respectively)7.SN itself did not effect the level of ROS in podocytes,while 10-8~10-4M SN was able to decrease the level of ROS induced by Ang ?(P<0.05 and P<0.01 respectively).8.Ang ? promoted p47-phox subunit expression in cytomembrane,and 10-8~10-4M SN was able to inhibite this effect(P<0.001).ConclusionWe found that a large number of high purity NETs-prone neutrophils could be elicited by garose gel subcutaneously implanted in mouse,and NETs promoted Ang ? secreted by podocytes.Ang ? promoted podocytes autophagy through ROS pathway via increased p47-phox expression in cytomembrane,and SN was able to inhibit this effect.Our findings indicated the probable mechanism of podocyte injured with NETs.