Knockdown Of NUP160 Impairs Mouse Podocytes

Primary nephrotic syndrome（PNS）is a common glomerular disease in children,accounting for approximately 21% to 31% of hospitalized children with urinary system diseases.The main clinical manifestations of PNS include massive proteinuria,hypoproteinemia,hyperlipidemia and edema.According to its response to steroid therapy,PNS is classified into steroid-sensitive nephrotic syndrome（SSNS）and steroid-resistant nephrotic syndrome（SRNS）categories.The prognosis of SRNS is poor.Fifty percent of patients gradually progress to end-stage renal disease（ESRD）in 5 years after diagnosing with SRNS.With the rapid development of genetics and gene detection technologies,the research on the pathogenesis of SRNS has attracted more and more attention to the podocyte injure caused by genetic factors.Podocytes,also known as glomerular visceral epithelial cells,form the glomerular filtration barrier together with endothelial cells and the glomerular basement membrane（GBM）.The foot processes of neighboring podocytes are interdigitated to form the slit diaphragm,which is the final barrier to filtration of plasma protein.Podocytes synthesize the components of GBM and maintain the normal morphology of GBM.Podocytes support glomerular capillaries,counteract capillary hydrostatic pressure and stabilize glomerular capillary network.To date,more than 50 monogenic genes have been discovered to cause podocyte injure when mutated.These include genes encoding slit diaphragm constituting molecules,such as NPHS1,NPHS2,CD2 AP,genes encoding the actin cytoskeleton,such as ACTN4,INF2,MYO1 E,genes encoding actin-regulating small GTPases of the Rho/Rac/Cdc42 family,such as ARHGDIA,ARHGAP24,KANK,and genes encoding nucleoporins,such as NUP93,NUP107 and NUP205,among others.NPHS1,NPHS2 and CD2 AP are the coding genes of nephrin,podocin and CD2-associated protein（CD2AP）,which are critical for maintaining the structural integrity of the slit diaphragm,signaling transduction in podocytes and the arrangement of the podocyte cytoskeleton.Changes in the expression and distribution of nephrin,podocin and CD2 AP disrupt the glomerular filtration barrier,leading to increased glomerular permeability and proteinuria.It has been found that NPHS1 mutations can lead to congenital nephrotic syndrome,and NPHS2 mutations can lead to early-onset autosomal recessive SRNS,and CD2 AP mutations can lead to early-onset focal segmental glomerulosclerosis（FSGS）.ACTN4 is the coding gene of α-actinin-4,which plays a role in connecting actin filaments with the slit diaphragm complex in podocytes.Changes in the expression and function of α-actinin-4 can cause depolymerization or redistribution of actin microfilaments and promote podocyte foot process effacement.ACTN4 mutations can cause autosomal dominant FSGS.ARHGDIA is the coding gene of ARHGDIA,which combines with Rho GDPase to prevent GDP dissociation and inhibit Rho-protease activation.Excessive activation of Rho-protease can lead to abnormal cell proliferation,apoptosis and migration.ARHGDIA mutations can cause early-onset SRNS.NUP93,NUP107 and NUP205 are the coding genes of nucleoporin 93kDa（Nup93）,nucleoporin 107kDa（Nup107）and nucleoporin 205kDa（Nup205）,which are components of the nuclear pore complexs.Nucleoporins（Nups）are mainly responsible for nucleocytoplasmic transport in eukaryotic cells.Beyond this vital role,Nups can regulate the transcriptional activity of specific target genes in a transport-independent manner.Nups can associate with specific genes and regulate their expression by controlling the chromatin structure and accessibility of transcription factors.Nup-mediated gene expression regulation is also reflected by the role of Nups in the establishment of chromatin boundaries and in transcriptional memory.Additionally,in proliferative cells,Nups play an important role in genome integrity maintenance and mitotic progression.Nups facilitate the repair of a subset of persistent DNA lesions through interactions with the DNA repair machinery.In mitotic cells,Nups assist nuclear envelope breakdown and centrosome migration.By ensuring the localization and function of key mitotic components,Nups promote accurate spindle assembly,mitotic progression,and faithful chromosome segregation.Changes in the structure and function of Nups can cause abnormal cell cycle,gene expression and signal transduction.Mutations in NUP93 inhibit podocyte proliferation and promote the apoptosis of podocytes and lead to early-onset SRNS.Mutations in NUP107 cause hypoplastic glomerulus structures and abnormal podocyte foot processes and lead to early-onset SRNS.Although more and more monogenic genes have been discovered to cause SRNS when mutated,the etiology of 80% of SRNS is unknown.Previously,we detected compound heterozygous mutations of the NUP160 gene（NC₀00011.10）（c.2407G>A,p.Glu803 Lys and c.3517C>T,p.Arg1173*）by whole-exome sequencing in a Chinese family with SRNS.Bioinformatics analysis predicts that the mutations of NUP160（c.2407G>A,p.Glu803 Lys and c.3517C>T,p.Arg1173*）are pathogenic mutations,suggesting a possible role for NUP160 in podocytes,and NUP160 gene may be a hitherto unrecognized monogenic cause of familial SRNS.NUP160 gene（NC₀00011.10）maps to chromosome 11 p and consists of 36 exons,whose transcription product NUP160 mRNA（NM₀15231）contains 5383 nucleotides,of which the number of coding nucleotides is 4311,from the 86 th nucleotide to the 4396 th nucleotide.NUP160 gene is highly expressed in HEK293 cells,HeLa cells and B-lymphoblasts,and also expressed in various tissues and organs such as kidney,heart,liver,hypothalamus,bone marrow and lymph nodes.NUP160 gene encodes Nucleoporin 160kDa（Nup160）（NP₀56046.1）,which contains 1436 amino acids.Nup160 is an important component of the scaffold of nuclear pore complexe（NPC）.In vertebrates,Nup160,Nup107,Nup85,Nup133,Nup96,Sec13,Nup37,Nup43,ELYS,and Seh1 form Nup107-160 subcomplex,playing an important role in chromosome segregation and NPC assembly in eukaryotes.In mitosis,the Nup107-160 complex is recruited to kinetochores and promotes spindle assembly.After mitosis,the Nup107-160 complex recruits other Nups or subcomplexes to reform NPCs.In-vitro experiments showed that depletion of the Nup107-160 complex emerged defects in spindle assembly and delayed mitosis,and formed continuous nuclear membrane without NPCs.Nup160,Seh1 and MOS3（a homolog of Nup96）is critical for RNA export in arabidopsis and is required for plant growth,flowering,pathogen defense and plant tolerance to cold stress.Nup160 and Nup96 are involved in the reproductive isolation,hybrid incompatibilities and female infertility caused by Drosophila simulans and Drosophila melanogaster hybridization.In human cells,down-regulation of the Nup107-160 complex caused DNA damage accumulation.The distribution of Nup160,Ndc1,Nup153 and Nup93 is abnormal in dilated and ischemic cardiomyopathy.In the SRNS family of our previous study,the proband presented with non-syndromic SRNS at age 7.Renal biopsy showed FSGS,and electron microscopy showed diffuse podocyte foot process effacement.The proband progressed to ESRD at age 15 and underwent renal transplant at age 16.4 and has experienced no recurrence of nephrotic syndrome（NS）to date.The pathogenic mutation of NUP160（c.2407G>A,p.Glu803Lys）is maternal origin and the pathogenic mutation of NUP160（c.3517C>T,p.Arg1173*）is paternal origin.Therefore,the mutations of NUP160（c.2407G>A,p.Glu803 Lys and c.3517C>T,p.Arg1173*）are compound heterozygous mutations,which is consistent with the genetic form of the SRNS family,i.e.autosomal recessive inheritance and can explain the etiology of the proband.Because the ultrastructure of the proband’s renal tissue showed diffuse podocyte foot process effacement,we speculated that the mutations of NUP160（2407G>A and 3517C>T）caused the loss of function of NUP160,resulting in podocyte injure and dysfunction.ObjectiveWe explore whether loss of NUP160 impairs podocytes by observing the effect of NUP160 knockdown on the mouse podocyte proliferation,apoptosis,autophagy and migration ability and investigating the effect of NUP160 knockdown on the expression of podocyte associated molecules including nephrin,podocin,CD2 AP and α-actinin-4.Methods1.We used the replication-incompetent lentiviral vector pLKO.1 chosen by the RNAi Consortium（TRC）for cloning and expressing shRNA sequences.The coding sequences of Mus musculus NUP160 mRNA（NM₀21521）were searched for the interference target sites.The forward and reverse oligonucleotides were annealed,and the resulted double strand DNA was ligated into the pLKO.1 digested with EcoR I and Age I.After being confirmed the presence of the shRNA inserted by digesting with EcoR I and Nco I and further being verified by sequencing with pLKO.1 sequencing primer,the successfully constructed recombinant plasmid pLKO.1-NUP160_shRNA,packaging plasmids psPAX2 and envelope plasmid pMD2.G were co-transfected into HEK293 T cells using PEI.The lentivirus-containing supernatant was harvested and was added into conditionally immortalized mouse podocytes.After 24 h,the virus-infected cells were selected with puromycin（2 μg/ml）for an additional 10 days to establish sublines of mouse podocytes with stable knockdown of NUP160.The efficiency of NUP160 shRNA transfection was confirmed by quantitative real-time polymerase chain reaction（qRT-PCR）,Western Blot and immunofluorescence assay.2.To investigate the effect of NUP160 knockdown on podocyte proliferation,CCK-8 assay was performed and optical density（OD）was measured under 450 nm for plotting cell proliferation curve.To investigate the effect of NUP160 knockdown on cell cycle,Propidiumiodide（PI）staining combined with flow cytometry（PI-FCM）was performed.Furthermore,the expression of CCND1（cyclin D1）,CDK4（CDK4）and CDKN1B（p27）was examined by qRT-PCR and Western Blot.3.To investigate the effect of NUP160 knockdown on cell apoptosis,Annexin V-FITC/PI staining combined with flow cytometry was adopted.The expression of Caspase-3 and PARP was examined by Western Blot.4.To investigate the effect of NUP160 knockdown on podocyte autophagy,the mRNA and protein expression of the autophagy-related genes BECN1（Beclin1）and LC3B（LC3B）was examined using qRT-PCR,Western Blot or immunofluorscence assay.5.To investigate the effect of NUP160 knockdown on the migration ability of podocytes,cell wound-healing assay and transwell assay were performed.6.To investigate the effect of NUP160 knockdown on podocyte associated molecules,the expression of NPHS1（nephrin）,NPHS2（podocin）,CD2AP（CD2AP）and ACTN4（α-actinin-4）at mRNA and protein levels and the subcellular localization of them was examined by qRT-PCR,Western Blot and immunofluorscence assay.All assays were performed at least three times.Statistical analyses were performed using SPSS 18.0 software.Quantitative data are presented as the means ± standard deviation.Statistical evaluation was performed using one-way analysis of variance,followed by least significant difference（LSD）or Dunnett’s T3 for the post hoc test between two groups.Results1.The efficiency of NUP160 shRNA transfection was confirmed by qRT-PCR,Western Blot and immunofluorescence assay.The results showed that all three NUP160 shRNA lentivirus vectors decreased the expression of NUP160 mRNA and Nup160 protein.The level of NUP160 mRNA was inhibited by 49.43%,74.56% and 62.02% in podocytes transfected with NUP160-shRNA-1,NUP160-shRNA-2 and NUP160-shRNA-3,respectively.Nup160 expression was inhibited by 39.28%,68.66% and 57.78% in podocytes transfected with NUP160-shRNA-1,NUP160-shRNA-2 and NUP160-shRNA-3,respectively.NUP160-shRNA-2 was selected for further study due to the highest efficiency of inhibition.2.OD values from podocytes stably transfected with NUP160 shRNA were significantly lower than those of either the normal or negative controls at 24 h,48 h,72 h and 96 h time points（all of P < 0.001）.The proportion of cells in the G0/G1 phase in podocytes stably transfected with NUP160 shRNA was significantly increased,while that in the S phase was significantly decreased（both of P < 0.001）.Furthermore,both mRNA and protein levels of CCND1（cyclin D1）and CDK4（CDK4）were decreased in podocytes stably transfected with NUP160 shRNA（P < 0.001 for CCND1 mRNA,CDK4 mRNA and Cyclin D1,P < 0.01 for CDK4）,while those of CDKN1B（p27）were increased（both of P < 0.001）.3.The podocytes stably transfected with NUP160 shRNA had a higher apoptotic rate.The percentage of early apoptotic cells and the percentage of late apoptotic cells in podocytes stably transfected with NUP160 shRNA were significantly higher than those either in normal or negative controls（both of P < 0.001）.Consistent with this result,western blot demonstrated that the levels of cleaved-Caspase-3 and cleaved-poly（ADP-ribosyl）transferase（cleaved-PARP）in podocytes stably transfected with NUP160 shRNA were significantly higher than that in either normal or negative controls（both of P < 0.001）.4.The mRNA levels of the autophagy-related genes BECN1 and LC3 B increased after NUP160 knockdown（both of P < 0.001）.Beclin1 protein expression and conversion of the cytosolic form of LC3 B I to its lipidated membrane-bound form LC3 B II also increased after NUP160 knockdown（P < 0.01 for Beclin1,P < 0.001 for LC3 B II / LC3 B I）.Similar results were observed by immunofluorescence assay（P < 0.001）.5.The data from the cell wound-healing assay showed that the wound gap between the podocytes with stable knockdown of NUP160 was significantly narrower than that in either normal or negative controls.The wound-healing rate of the podocytes with stable knockdown of NUP160 was significantly higher than that of the two control groups（P < 0.001）.The transwell assay showed that the number of cells passing through the chamber membrane in the group with stable knockdown of NUP160 was significantly greater than that of the two control groups（P < 0.001）.6.Upon Nup160 suppression,the expression of NPHS1（nephrin）,NPHS2（podocin）and CD2AP（CD2AP）were decreased at both the mRNA（P < 0.05,< 0.001 and < 0.001,respectively）and protein levels（P < 0.05,< 0.001 and < 0.001,respectively）,and ACTN4（α-actinin-4）was increased（P < 0.001 for ACTN4 and P < 0.01 for α-actinin-4,vs.the normal control group;P < 0.01 for ACTN4 and P < 0.05 for α-actinin-4,vs.the negative control group）.Similar results were observed by immunofluorescence assay（P < 0.05,< 0.001,< 0.01 and < 0.001,respectively,vs.the normal control group;P < 0.05,< 0.001,< 0.05 and < 0.001,respectively,vs.the negative control group）.In addition,in both normal control and negative control,nephrin and podocin showed a perinuclear and membrane staining pattern,but in the podocytes with stable knockdown of NUP160,the membrane surface staining of nephrin and podocin decreased evidently and was mainly localized around nuclei.In both normal control and negative control,CD2 AP was diffuse in the cytoplasm and on the membrane,but in the podocytes with stable knockdown of NUP160,the cytoplasm staining of CD2 AP decreased evidently and was mainly localized around nuclei.The subcellular localization of a-actinin-4 was alike in normal control,negative control and NUP160 knockdown group,all of which showed cytoplasmic filamentous staining extending to the processes.ConclusionsThe knockdown of NUP160 impaired mouse podocytes,i.e.the knockdown of NUP160 significantly inhibited the proliferation of podocytes,and promoted the apoptosis of podocytes,and induced the autophagy of podocytes and enhanced cell migration,and the knockdown of NUP160 also altered the expression and localization of podocyte associated molecules,including nephrin,podocin,CD2 AP and α-actinin-4.