The Mechanism Of LncPvt1 Regulating Autophagy Level In Homocysteine Induced Mouse Podocyte Injury

Objective To observe the effect of homocysteine?Hcy?on the expression of lncRNA Pvt1?lncPvt1?,to determine whether lncPvt1 can regulate the level of autophagy,to determine the effect of the change of autophagy level on the damage of podocytes,and to elucidate the mechanism of lncPvt1 regulating the level of autophagy and promoting the damage of glomerular podocytes induced by homocysteine.Methods Animal model replication:nine wild-type male C57BL/6J mice(CBS^+/+)were used as the control group,and nine male C57BL/6J mice(CBS^+/-)as the model group.The two groups were fed with high methionine feed for 4 weeks.The glomerular morphology was observed by PAS staining,the ratio of PAS positive area to total glomerular area was counted,and the changes of podocyte damage and autophagosome were observed by transmission electron microscope.Western blot was used to detect the expression of podocyte marker proteins Nephrin,Podocin and autophagy related proteins LC3,p62 in renal cortex.Immunofluorescence staining was used to observe the expression of LC3 and p62 in glomeruli.The expression of lncPvt1 in renal cortex was detected by qRT-PCR,and the expression of lncPvt1 in glomerulus was observed by fluorescence in situ hybridization?FISH?.In vitro cell model replication,different Hcy concentrations?0?mmol/L,20?mmol/L,40?mmol/L,60?mmol/L,80?mmol/L,100?mmol/L?were used to stimulate podocytes for 12h,24h and 48h,and the most significant concentration and time of increased autophagy level of podocytes were screened?the experimental results showed that 80?mmol/L,24h?.According to the screening results,podocytes were divided into two groups:Control group?treated with 0?mmol/L Hcy for 24h?,Hcy group?treated with 80?mmol/L Hcy for 24 h?.Detection of lncPvt1 expression and nucleocytoplasmic distribution in podocytes by qRT-PCR and FISH.After transfection of podocytes by mRFP-GFP-LC3 adenovirus,autophagy inhibitor was used to inhibit Hcy interference in glomerular podocytes,and the changes of autophagy flux were observed.At the same time,lncPvt1 overexpression/interference vector was constructed and transfected into podocyte,and qRT-PCR verified its own lncPvt1 expression;western blot and immunofluorescence were used to detect the expression of autophagy-related proteins LC3 and p62 after Hcy intervention.Results1.The glomerular morphology was observed by PAS staining,and the ratio of the positive area of PAS staining to the total area of glomerulus was calculated:compared with the Cbs^+/+group,the proliferation of mesangial cells and mesangial matrix increased,the mesangial area widened,and the ratio of PAS staining positive area to total glomerular area increased in the Cbs^+/-group?P<0.05?.2.Western blot was used to detect the expression of Nephrin and Podocin protein.The results showed that:compared with the Cbs^+/+group,the expression of Nephrin and Podocin protein in the Cbs^+/-group was decreased?P<0.05?.3.Observation of glomerular podocytes by transmission electron microscope:compared with the CBS^+/+group,the injury of the Cbs^+/-group was obvious,and the level of autophagy was increased.4.Detection of LC3 and p62 protein expression in renal cortex by Western blot and immunofluorescence,The results showed that:compared with the Cbs^+/+group,LC3II/I increased?P<0.05?and p62/?-actin decreased?P<0.05?in the Cbs^+/-group,and the results of immunofluorescence were consistent with those of Western blot.5.Screening the concentration and time of Hcy which is the most significant increase in podocyte autophagy,the results showed that:compared with the 0?mol/L 24h group,the LC3II/I value increased most significantly?P<0.05?and the p62/?-actin decreased most significantly?P<0.05?in the 80 mol/L 24h group.6.Autophagic flux assay showed that the level of autophagy increased in Hcy group compared with that in Control group,and decreased in Hcy+3MA group compared with Hcy group.7.The expression of lncPvt1 in renal cortex was detected by qRT-PCR,the results showed that:compared with the CBS^+/+group,the expression of lncPvt1 was increased in the Cbs^+/-group?P<0.05?;compared with the Control group,the expression of lncPvt1 was significantly increased in the Hcy group?P<0.05?.FISH detected glomerular lncPvt1expression:compared with the CBS^+/+group,the expression of glomerular lncPvt1 was increased in the Cbs^+/-group.8.qRT-PCR and FISH were used to detect the nuclear-cytoplasmic expression distribution of lncPvt1 in Hcy group,and the results showed that:lncPvt1 was distributed in both cytoplasm and nucleus.9.Construction of lncPvt1 overexpression/interference vector and transfection of podocytes,qRT-PCR validated the expression of lncPvt1 in podocytes after transfection:compared with Lv-GFP group,the expression of lncPvt1 in Lv-Pvt1 group increased?P<0.05?;compared with ASO-NC group,the expression of lncPvt1 in ASO-Pvt1 group decreased?P<0.05?.10.Western blot and immunofluorescence were used to detect LC3 and p62 protein in podocytes of each group after Hcy intervention.The results showed that compared with Lv-GFP+Hcy group,LC3II/I of Lv-Pvt1+Hcy group increased?P<0.05?,p62/?-actin decreased?P<0.05?;compared with ASO-NC+Hcy group,LC3II/I of ASO-Pvt1+Hcy group decreased?P<0.05?,p62/?-actin increased?P<0.05?.The results of immunofluorescence were consistent with those of Western blot.Conclusion One of the possible mechanisms of Hcy induced glomerular podocyte injury is that Hcy up-regulates the expression of lncPvt1,which leads to the increase of autophagy level of glomerular podocytes.