| Lupus nephrits(LN)is one of the most sever and frequent complications of systemic lupus erythematosus(SLE).Long-term severe proteinuria is one of the main clinical manifestation of LN,and a sign of poor prognosis.In a variety of distinct mechanisms of proteinuria,the podocytes dysfunction or injury is the most important cause.Therefore,it is of great significance and urgency to explore the specific mechanism of podocyte injury in LN,thereby further preventing proteinuria and relieving renal damage in LN patients.In this study,the kidney tissue of LN patients,MRL/lpr lupus-prone mice,and mouse podocyte lines(MPCs)were used as the study objects to investigate the role and possible mechanism of nestin in podocyte injury and proteinuria formation in LN,which will provide a basis for further elucidating the pathogenesis of LN and its targeted therapy.The experiment is divided into the following four parts: Part One Nestin contributed to the proteinuria formation by regulating nephrin in lupus nephritisObjective:In this part,we explore the role and possible mechanism of nestin in podocyte injury and proteinuria formation in LN.Methods:1.The renal biopsy of LN patients and the control renal tissues,which were obtained from patients with renal tumors during operation,and pathologically diagnosed as normal kidney tissue.According to the mean proteinuria the LN patients were divided to two groups: mild proteinuria group(LN-MP)and severe proteinuria group(LN-SP).The expression of nestin,synaptopodin and nephrin was detected by immunohistochemistry(IHC)and immunofluorescence(IF).2.The female MRL/lpr lupus-prone mice aged 30 weeks were divided into two groups according to the level of proteinuria: mild proteinuria MRL/lpr mice(MRL/lpr-MP,n=10)and severe proteinuria MRL/lpr mice(MRL/lpr-SP,n=10).Ten MRL/MPJ mice,whose age and sex matched to the MRL/lpr mice,served as control group.The 24 h proteinuria was detected by a mouse urine protein enzyme-linked immunosorbent assay quantitation kit.And the expression of nestin,nephrin and p-nephrin(Y1217)in renal cortex was detected by IHC and Western blot assay.3.The differentiated mature MPCs were used to detecte the effect of nestin on the expression and phosphorylation of nephrin in LN.⑴ To investigate the effect of LN plasma on the expression of nestin,nephrin and p-nephrin(Y1217),the MPCs were randomly exposed to LN plasma(10%)and collected at 0 h,12 h,24 h,36 h and 48 h.And Western blot was used to test the protein level of nestin,nephrin and p-nephrin(Y1217)in MPCs.⑵ To further explore the role of nestin in expression and phosphorylation of nephrin,the MPCs were stimulated with control plasma or LN plasma after knocking down or overexpressing nestin.The expression of nestin,nephrin and p-nephrin(Y1217)was detected by Western blot.Results:1.The expression of nestin and nephrin in podocytes of LN was positively correlated,and both were negatively correlated with proteinuria.⑴ IF showed that the expression of nestin increased in podocytes in LN-MP group compared with the control group.And a notable decrease in nestin was observed in LN-SP group compared with LN-MP group.IHC showed that the nephrin was significantly correlated with nestin in the glomeruli of LN patients.Specifically,nephrin expression was decreased with lower nestin in LN Ⅳ and LN Ⅴ patients with aggravated proteinuria.Moreover there was a significantly negative correlation between proteinuria and nestin or nephrin.⑵ IHC showed that the nephrin was significantly correlated with nestin in the same glomeruli of MRL/lpr mice.Moreover,there was a significantly negative correlation between proteinuria and nestin or nephrin.Western blot assay showed that nestin,nephrin and p-nephrin(Y1217)expression significantly increased in MRL/lpr-MP mice compared with MRL/MPJ mice,while significantly decreased in MRL/lpr-SP mice compared with MRL/lpr-MP mice.2.Nestin could regulate the expression and phosphorylation of nephrin in LN⑴ LN plasma could upregulate the expression of nestin,nephrin and p-nephrin(Y1217)in MPCs.Western blot assay showed nestin,nephrin and p-nephrin(Y1217)expression increased in MPCs stimulated by LN plasma at 24 h,and then decreased.⑵ Nestin could regulate the expression and phosphorylation of nephrin in MPCs.Western blot assay showed that knockdown of nestin significantly decreased the nephrin and p-nephrin expression in MPCs.After transfection with WT-Nestin plasmid,the protein level of nestin was significantly elevated as compare with NC group.Conclusion:Nestin could regulate the expression and phosphorylation of nephrin in podocyte of LN,and participate in the formation of proteinuria.Part Two Nestin regulated the expression and phosphorylation of nephrin by enhancing the mitophagy in LNObjective:In this part,we investigate the role and possible mechanism of nestin and mitophagy in podocyte injury and proteinuria formation in LN.Methods:1.The clinical and animal specimens were collected.And the expression of nestin and LC3,p62 was detected by IF.The ultrastructure of the podocytes in renal cortex of MRL/lpr mice was observed with a transmission electron microscopy(TEM).And the expression of nestin,LC3 and p62 was detected by IF.2.The differentiated mature MPCs were used to detecte the relationship between the nestin and autophagy and the effect of them on podocyte injury in LN.⑴ To investigate the effect of LN plasma on autophagy,the MPCs were randomly exposed to LN plasma(10%)and collected at 0 h,12 h,24 h,36 h and 48 h.And the m RFP-GFP-LC3 plasmid was used to measure the autophagy flux.Western blot and IF technique were used to test the protein level of atg5,atg12,atg16,LC3,p62 and PINK1.⑵ To detect the effect of autophagy inhibition on the expression of nestin,nephrin and p-nephrin(Y1217)in podocyte of LN,the MPCs were randomly divided into five groups: control plasma,LN plasma,LN plasma+si PINK1,LN plasma+si NC and LN plasma+3-MA group.The expression of atg5,atg12,atg16,LC3,p62,PINK1,nestin,nephrin and p-nephrin(Y1217)was detected by Western blot,⑶ To further explore the role of nestin in autophagy in podocyte of LN,the MPCs were divided as the same as the method in part one.The expression of nestin,atg5,atg12,atg16,LC3,p62 and PINK1 was detected by Western blot.Results:1.The autophagy levels increased in podocytes of LN with mild proteinuria.⑴ It has found a large number of autophagosomes in the podocytes with slight foot fusion in MRL/lpr-MP mice,but few autophagosomes in the podocytes with severe foot fusion in MRL/lpr-SP mice using TEM.⑵ IF showed that the nestin and LC3 expression both increased and p62 decreased in the MRL/lpr-MP mice compared with MRL/MPJ group.Nevertheless,nestin and LC3 decreased and p62 increased in the MRL/lpr-SP mice compared with the MRL/lpr-MP mice.IF also showed that the nestin and LC3 expression both increased and p62 decreased in the LN-MP patients compared with control group.Nevertheless,nestin and LC3 decreased and p62 increased in the LN-SP group compared with the LN-MP group.2.Nestin regulated the expression and phosphorylation of nephrin by enhancing the mitophagy in LN.⑴ LN plasma could upregulate the autophagy associated proteins in MPCs.The m RFP-GFP-LC3 plasmid showed the autophagy flux was enhanced in MPCs stimulated by LN plasma at 24 h.And the IF and Western blot assay showed the atg5,atg12,atg16,LC3,p62 and PINK1 expression increased in MPCs stimulated by LN plasma at 24 h.⑵ The inhibition of autophagy could downregulate the expression and phosphorylation of nephrin in podocyte.Western blot assay showed that the 3-MA significantly decreased nephrin and p-nephrin(Y1217)expression induced by LN plasma in MPCs.And knockdown of PINK1 expression also downregulated nephrin and p-nephrin(Y1217)expression induced by LN plasma.However,nestin were not affected by 3-MA and si PINK1.⑶ Nestin could regulate the autophagy level in podocyte.Western blot assay showed that knockdown of nestin significantly decreased the atg5,atg12,atg16,PINK1 and LC3 protein expression,and increased the p62 expression induced by LN plasma in MPCs.After transfection with WT-Nestin plasmid,the protein level of atg5,atg12,atg16,PINK1 and LC3 was significantly elevated,and p62 was decreased.Conclusion:Nestin regulated the expression and phosphorylation of nephrin by enhancing the mitophagy in LN.Part Three Nestin regulated the expression and phosphorylation of nephrin by oxidative stress in LNObjective:In this part,we investigate the role and possible mechanism of oxidative stress in podocyte injury in LN.Methods:The differentiated mature MPCs were used to detect the relationship among the nestin,autophagy and oxidative stress,and the effect of them on podocyte injury in LN.⑴ To investigate the effect of LN plasma on ROS,and the expression of PTP1 B and fyn,the MPCs were randomly exposed to LN plasma(10%)and collected at 0 h,12 h,24 h,36 h and 48 h.And the Mito SOX staining and flow cytometry(FCM)were used to detect the ROS production.Western blot was used to test the protein level of PTP1 B and fyn.⑵ To further investigate the effect of N-acetyl cysteine(NAC)and Mito-TEMPO on nestin,mitophagy,nephrin and p-nephrin(Y1217),the MPCs were randomly divided into five groups: control plasma,LN plasma,LN plasma+NAC,LN plasma+Mito-TEMPO,LN plasma+DMSO group.And Western blot was used to detect the expression of nestin,nephrin,p-nephrin(Y1217),LC3,p62,PINK1 and PTP1 B protein.⑶ To detect the effect of autophagy inhibition on ROS production and PTP1 B expression in podocyte of LN,the MPCs were divided as the same as the method in part two.Mito SOX staining and FCM were used to detect the ROS production.Western blot was used to test the protein level of PTP1 B.⑷ To further explore the role of nestin in ROS production,PTP1 B and fyn expression in podocyte of LN,the MPCs were divided as the same as the method in part one.Mito SOX staining and FCM were used to detecte the ROS production.Western blot was used to test the protein level of PTP1 B and fyn.⑸ To investigate whether the inhibition of PTP1 B or fyn could regulate the phosphorylation of nephrin,we randomly divided the MPCs into five groups: control plasma,LN plasma,LN plasma+PTP1B inhibitor,LN plasma+SU6656,and LN plasma+DMSO group.Western blot was used to detect the expression of PTP1 B,fyn,nephrin and p-nephrin(Y1217).Results:1.Mitophagy and ROS production induced by LN plasma interacted with each other,and were regulated by nestin.⑴ LN plasma could affect the ROS production in MPCs.Mito SOX and FCM showed that ROS production was decreased in MPCs stimulated with LN plasma at 24 h and increased at 48 h compared to 0 h group⑵ NAC and Mito-TEMPO could downregulate the autophagy induced by LN plasma in MPCs.Western blot showed that the PINK1 and LC3 expression decreased and the p62 increased in MPCs pretreated with NAC and Mito-TEMPO and stimulated with LN plasma.⑶ The inhibition of autophagy could downregulate the ROS production induced by LN plasma in MPCs.Mito SOX and FCM showed that ROS production was decreased in MPCs pretreated with 3-MA.⑷ Nestin could regulate the ROS production in MPCs.Mito SOX and FCM showed that the ROS generation was much lower once knocking down the nestin expression,and increased due to overexpression of nestin.2.Nestin could mediate PTP1B/fyn balance by regulating the level of oxidative stress in podocytes of LN,thus affecting the expression and phosphorylation level of nephrin protein.⑴ LN plasma could upregulate the expression of PTP1 B and fyn in MPCs.Western blot assay showed the expression of PTP1 B and fyn increased in MPCs stimulated by LN plasma at 24 h.⑵ The inhibition of PTP1 B and fyn could upregulate or downregulate the expression and phosphorylation of nephrin in podocyte.Western blot showed PTP1 B inhibitor promoted the expression and phosphorylation of nephrin,however,was abolished by SU6656 in MPCs stimulated with LN plasma.⑶ NAC and Mito-TEMPO could upregulate the expression of PTP1 B,and downregulate nephrin and p-nephrin(Y1217)expression in MPCs stimulated with LN plasma.Western blot showed that the PTP1 B expression increased,and nephrin and p-nephrin(Y1217)decreased in the MPCs pretreated with NAC and Mito-TEMPO and stimulated by LN plasma.⑷ The inhibition of autophagy could upregulate the PTP1 B expression in MPCs stimulated with LN plasma.Western blot assay showed that the 3-MA significantly increased the PTP1 B expression induced by LN plasma in MPCs.And knockdown of PINK1 expression also upregulated PTP1 B expression induced by LN plasma.⑸ Knocking down nestin expression could upregulate the expression of PTP1 B and downregulate fyn expression in podocyte stimulated with LN plasma.Western blot assay showed that knockdown of nestin significantly decreased the fyn expression,and increased the PTP1 B expression in MPCs.Conclusion:Nestin could break PTP1B/fyn balance by regulating the level of oxidative stress in podocytes of LN,thus affecting the expression and phosphorylation level of nephrin protein.Part Four Knocking down nestin expression aggravated podocyte injury and proteinuria in MRL/lpr miceObjective:In this part,the MRL/lpr lupus-prone mice were used as the study objects.Local infection technology was used to knock down the expression of nestin in the kidney of MRL/lpr mice,and to explore the role and possible mechanism of nestin in podocyte injury and proteinuria formation in LN.Methods:18 MRL/lpr mice aged at 27 weeks were randomly divided into three groups: MRL/lpr group,MRL/lpr-sh NC-Ad group,and MRL/lpr-sh Nestin-Ad group.Six MRL/MPJ mice,whose age and sex matched to the MRL/lpr mice,served as control group.The mice in MRL/lpr-sh NC-Ad group,and MRL/lpr-sh Nestin-Ad group were renally injected with adenoviruses in both kidneys.After three weeks,the mice were sacrificed at 30-week old after collecting the 24 h urine and blood samples,and the renal cortex was collected for relevant investigations.IF and real-time PCR were used to detect the silencing effect.The 24 h proteinuria was detected by a mouse urine protein enzyme-linked immunosorbent assay quantitation kit.TEM,HE and PAS staining were applied to detect the pathological changes in the kidneys of MRL/lpr mice.In addition,the expression of nestin,nephrin,p-nephrin(Y1217),LC3 and p62 was detected by IHC and Western blot assay.Results:1.Adenovirus infection technique could effectively knock down the expression of nestin in the renal cortex of MRL/lpr mice.The IF results demostrated that the positive signals were observed in the renal cortex after the adenovirus injection.And the real-time PCR indicated that the nestin m RNA level was significantly reduced in MRL/lpr-sh Nestin-Ad group.2.Knocking down nestin expression aggravated podocyte injury and proteinuria in MRL/lpr mice.⑴ ELISA showed that the proteinuria increased in MRL/lpr mice,and knockdown of nestin expression in podocytes could aggravate proteinuria in MRL/lpr mice.⑵ HE and PAS staining showed the larger renal glomeruli volume,mesangial cell expansion,and matrix accumulation in the MRL/lpr mice compared with MRL/MPJ mice.However,the pathological changes were strengthened by knockdown of nestin.⑶ TEM revealed that knockdown of nestin aggravated lesions and destroyed the integrity of podocytes by aggravating foot process fusion in MRL/lpr mice.3.Knocking down nestin could decreased the expression and phosphorylation of nephrin and autophagy level in MRL/lpr mice.⑴ IHC showed that the nestin,nephrin and LC3 expression was increased in MRL/lpr mice compared to MRL/MPJ mice,and the p62 decreased.However,the results were reversed after knockdown of nestin.⑵ Western blot showed that the nestin,nephrin and p-nephrin(Y1217)expression was increased in MRL/lpr mice compared to MRL/MPJ mice,the results were reversed after knockdown of nestin.Conclusion:Knocking down the nestin expression in kidney of MRL/lpr mice could downregulate the autophagy level and the expression of nephrin and p-nephrin(Y1217),and aggravated the proteinuria. |
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