Mechanism And The Protective Effect Of Autophagy On Podocytes In Lupus Nephritis

Background and objectivesSystemic lupus erythematosus(SLE)is a multisystem-involved autoimmune disease,while lupus nephritis is one of the most common and severe complication of SLE and a major influence factor on prognosis of SLE.About 50%-70%of SLEs would involve the kidney within 5 years after diagnosis,while 5%-15%of SLE patients finally develop to end-stage renal disease within 10 years.Since glucocorticoid combined with immunosuppressants has been used into lupus nephritis treatment,the complete response rate has been significantly improved,but the 6-month complete response rate is still below 10%and the 3-year complete response rate is only about 60%.Clearly,the prognosis of lupus nephritis is not optimistic Moreover,SLE mainly attacks women at the childbearing age,and lupus nephritis brings extreme suffering and economic burden to the society and families.Thus,the pathogenesis and new targeted therapy of lupus nephritis are extremely important and urgentDuring the biological process of autophagy,the lysosomes in membrane vesicles degrade the cytoplast contents,mainly the macromolecules of injured organelles and aging proteins.Autophagy provides the body the raw materials needed to synthesize new proteins and update organelles,thereby maintaining the protein metabolism balance and environmental stability in cells.Abnormal autophagy may cause tumors,cardiovascular diseases,liver disease and autoimmune diseasesStudies on the autophagy and SLE correlation show SLE patients possess higher T lymphocytes autophagy levels compared with healthy people or patients with non-SLE autoimmune diseases(vasculitis or sicca syndrome).Defect of the autophagy gene Atg5 will interfere with B cell development.The proliferation of specific CD4+T cells can be inhibited by using 3-methyladenine or wortmannin to block the autophagy pathway of mucoproteinl-transfected dendritic cells.It is believed autophagy may play important roles in the occurrence of SLE by regulating T lymphocytes,B lymphocytes,dendritic cells and macrophagesThe existing research on autophagy and SLE is focused on immune cells,but whether autophagy also regulates renal residential cells when SLE involves the kidney?Whether autophagy or whether abnormal autophagy is involved in the occurrence and development of lupus nephritis?At present,there are only some indirect evidences For instance,Hartleben et al.knocked out the autophagy-correlated gene Atg5 from mice and found albuminuria,glomerulus sclerosis,and loss of kidney podocytes.A German research team used the ds-DNA antibody isolated from the blood of SLE patients to irritate podocytes and found cytoplast antibody aggregated in podocytes,inducing the podocyte injuries,and the use of autophagy inhibitor led to intracellular antibody accumulation,indicating autophagy may be involved in regulating the antibody elimination and injuries of podocytesOn this background,the present study was designed to detect the expression of autophagosomes during the course of lupus nephritis(LN).We use various methods including electron microscopy,immunohistochemistry,western blot and qRT-PCR to confirm the expression tendency of podocyte autophagy in this disease;to further investigate the survival of podocytes,endoplasmic reticulum stress of cells and proteinuria in mice after intervention of autophagy,to assess the effect of autophagy on LN;and to determine the expression of marker proteins of autophagy and podocytes after blocking the TLRs/NF-κB pathway with MyD88-siRNA,the final objective is to reveal the pathways and regulatory mechanisms for autophagy activationMethods and results1.Expression Analysis of autophagy in renal tissues and podocytes of LNElectron microscopy,qRT-PCR and immunohistochemistry were used and it was found that the expression of LC3 and Beclin1(p<0.05)as well as SQSTM1/P62,an index of autophagy degradation,were increased in renal tissue and glomerular podocytes of LN compared with that of the normal kidney.Podocytes were treated with IgG from patients with LN and then treated with bafilomycin A1 or Atg5-siRNA,respectively,and the expression of related proteins was measured by Western blot,qRT-PCR and the tandem RFP-GFP-LC3 adenovirus construct.The results showed that the expression of LC3,Beclinl and p62 was increased at 24,48 and 72hrs,indicating that autophagy of podocytes was activated by autoantibodies and degradation disorder,however,was present in activated autophagy2.Analysis of the role of autophagy in podocyte injury during lupus nephritisPodocytes were incubated with LN-IgG,and then treated with rapamycin and Atg5-siRNA,respectively,for autophagy intervention.Western blot analysis showed the expression of podocyte markers including Nephrin and CD2AP decreased after LN IgG treatment;the expression of Nephrin and CD2APsignificantly decreased in podocytes when Atg5 was silenced;and in contrast,the expression of Nephrin and CD2AP was increased after rapamycin intervention compared with the LN IgG treatment group.Moreover,Cell apoptosis analysis indicated that Atg5-siRNA increased the percentage of early apoptosis and advanced apoptosis in podocytes However,apamycin decreased the percentage of early apoptosis and advanced apoptosis in podocytes.The results of animal experiment showed that the amount of urinary proteinsin of mice was significantly reduced after treatment with rapamycin and significantly increased after Atg5-siRNA intervention.Western blot analysis showed that compared with the LN group,the expression of Nephrin and CD2AP was increased and that of GRP94(an indicator for endoplasmic reticulum stress)was decreased in the rapamycin+LN group,and the expression of Nephrin and CD2AP was reduced and that of GRP94 was increased in the Atg5-siRNA+LN group.It was speculated that autophagy protected podocytes and inhibited the progression of LN.3.Analysis of the NF-κB signaling pathway mediating the activation of autophagy in LNWestern blot analysis of LN IgG-treated podocytes showed that the expression of MyD88 and nucleus NF-κB p65 was increased by Atg5-siRNA intervention,indicating autophagy could inhibit the NF-KB-associated inflammatory responses in LN.Subsequent Western blot analysis demonstrated that the expression of Nephrin and CD2AP in podocytes was increased after MyD88-siRNA intervention compared with that of the LN group,indicating that blocking MyD88 could reduce the antibody-induced podocyte injury.Finally,it was found that the expression of autophagy in antibody-induced podocytes injury was reduced after siRNA-MyD88 blocking,suggesting that activation of autophagy may be mediated by the TLRs/NF-κB signaling pathwayConclusionsThe increased autophagy in the kidney tissue of LN and the abnormally increased autophagy induced by LN IgG in podocytes contribute to the podocyte injury.Autophagy plays a role in protecting the podocytes and kidneys as evidenced by the changes in the podocytes survival status and urinary protein in LN mice after autophagy intervention.LN antibodies increase the endoplasmic reticulum stress in podocytes and autophagy protects podocytes by inhibiting the endoplasmic reticulum stress in these cells.In antibody-induced podocyte injury,autophagy may be activated by the MyD88-mediated TLRs/NF-κB signaling pathway,thereby negatively regulating TLRs/NF-κB-associated inflammatory responses to protect podocytes.The regulation of autophagy may serve as an effective approach for the treatment and prevention of LN in the future.