The Study On Mechanism Of Podocyte Injury Induced By Abp1 Overexpression And Whole Genome Association Study Of Childhood Steroid-sensitive Nephrotic Syndrome Without Relapse

PART ONE THE STUDY ON MECHANISM OF PODOCYTE INJURY INDUCED BY ABP1 OVEREXPRESSIONObjective: To explore the injury effects of podocyte injury induced by Abp1 overexpression and its mechanism.Methods: Section 1: the expression of Abp1 was detected by immunohistochemistry in 6 cases renal biopsies with minimal change(MCNS)and focal segmental glomerulosclerosis(FSGS),and simultaneous matching of normal renal tissue adjacent to Wilms tumor.In addition,20patients(10 MCNS and 10 FSGS)in the clinical information system of children’s Hospital Affiliated to Chongqing Medical University were selected for cross-sectional analysis of urinary protein,serum creatinine and blood urea nitrogen onset.Section 2: ADR model was established in6-week-old SPF male BALB/C wild-type(WT)and Abp1 gene knockout(Abp1KO)mice.By the twelfth week,HE and PAS staining,double kidney index evaluation,glomerular area calculation,glomerular PAS positive staining area evaluation,biochemical examination of serum albumin and urea nitrogen,and electron microscopy were performed Meanwhile,the random urinary protein creatinine ratio was used to determine the changes of proteinuria at 0,4,8 and 12 weeks after ADR modeling.Section 3:MPC5 podocyte lines were transfected with Abp1 lentivirus to construct stable ABP1 overexpression podocyte lines(OEAbp1),Abp1 knockdown podocyte lines(sh Abp1)and corresponding negative control groups.Podocyte marker molecules,cell ultrastructure,F-actin expression,podocyte adhesion function and podocyte migration function of OEAbp1 and shi Abp1 were detected,respectively.Section 4: podocytes from OEAbp1 and negative control groups were compared by RNA-seq,and then GO and KEGG analysis were enriched and analyzed.The protein interaction network(PPI)was obtained by using Cytoscape 3.5.1.The core genes of PPI network were obtained by using Cyto NCA and MCODE plug-ins,respectively.Finally,the RNA-seq data were verified by RT-q PCR.Results: Section 1: the expression of Abp1 in the glomeruli of PNS patients was significantly increased(P<0.05).Meanwhile,compared with the control group,the urinary protein onset in MCNS and FSGS groups was significantly increased(P<0.01).Compared with the control group,the blood urea nitrogen onset in FSGS group was significantly increased(P<0.05).Section 2: By the twelfth week,compared with wild-type mice,Abp1 knockout mice had partial remission in the aspects of luster of hair color,eating status,activity,mental status,weight gain.Besides,renal inflammatory cell infiltration was relieved,mesangial cell proliferation was mild,and FSGS formation was not observed.The kidney index of wild mice injected with adriamycin was significantly increased(P<0.01),while the glomerulus was significantly reduced(P<0.01)and mesangial area was significantly proliferated(P<0.01).There was no significant difference in blood urea nitrogen level between Abp1 knockout mice injected with adriamycin and control group(P>0.05).The levels of serum albumin in wild-type mice and mice injected with adriamycin were significantly decreased,but there was no significant change in Abp1 knockout mice(P<0.01).The wild-type mice injected with doxorubicin showed typical proteinuria changes,while the ratio of urinary protein/creatinine of Abp1 knockout mice injected with doxorubicin only increased at the 12 th week,which was slower than that of wild-type mice injected with doxorubicin at the same conditions(P<0.05).Additionally,transmission electron microscopy showed that the glomerular ultrastructure of Abp1 knockout mice was partially relieved the twelfth week.Section 3: RT q PCR showed that the m RNA levels of synaptopodin and nephrin,the markers of OEAbp1,were significantly decreased(P<0.01),but there was no significant change in sh Abp1(P>0.05).Scanning electron microscope showed that the podocyte body of OEAbp1 was thin,the podocyte process was incomplete,and the podocyte junction was decreased,while the morphology of shi Abp1 had no significant change.Fluorescence microscopy showed that the expression of F-actin in OEAbp1 podocytes decreased,and there was a phenomenon of skeleton polymerization.There was no significant change in the morphology of shi Abp1 during the same period(P>0.05).In addition,the adhesion function and migration function of OEAbp1 podocytes were significantly decreased compared with the negative control group(P<0.01);the adhesion function of shi Abp1 podocytes had no significant difference compared with the negative control group(P>0.05).Section 4: 1130 differentially expressed genes(DEGs)were obtained by analyzing RNA-seq expression profile,including 483up-regulated DEGs and 647 down regulated DEGs.The analysis of go and KEGG based on DEGs indicated that the up-regulated and down regulated enrichment signals were quite different.Cytoscape 3.5.1 revealed the PPI network includes 366 nodes and 1668 connections,and three important key gene action modules are obtained simultaneously.The top 15 core proteins are kininogen 1(KNG1),ribosomal protein s15a(RPS15A),ribosomal protein S3a(RPS3A1),ribosomal protein S13(RPS13),fibrinogen gamma chain(FGG),ubiquitin A-52 residue ribosomal protein fusion product 1(UBA52),complement C3(C3),Fau ubiquitin like protein and ribosomal protein S30(FAU),fibrinogen alpha chain(FGA),Serpin family member 1(SERPINA1A),fibronectin 1(FN1),ribosomal protein L30(RPL30),albumin(ALB),ribosomal protein S25(RPS25),α-2-hs-glycoprotein(AHSG).RT-q PCR test verified the reliability of RNA-seq data.Conclusion: Abp1 is highly expressed in the kidney tissues of PNS patients(MCNS and FSGS),which is involved in the occurrence and development of PNS renal damage.Overexpression of Abp1 can mediate a wide range of damage effects in podocytes,including podocyte morphological abnormalities,skeletal F-actin expression and morphological changes,cell adhesion and migration dysfunction.Abp1 knockout can effectively alleviate adriamycin induced renal pathological damage.Abp1 is expected to be a potential target for PNS therapy.The core protein interaction module mediated by Abp1 overexpression provides a direction for PNS research in the future.PART TWO WHOLE GENOME ASSOCIATION STUDY OF CHILDHOOD STEROID-SENSITIVE NEPHROTIC SYNDROME WITHOUT RELAPSEObjective: To study the specific susceptibility genes and loci of patients with steroid-sensitive nephrotic syndrome without relapse(SSNSWR)by genome wide association analysis(GWAS),and to reveal the molecular genetic mechanism of SSNSWR.Methods: The target genome regions of 1983 Chinese Han people(148 SSNSWR and 1835 healthy controls)were typed by the whole exon probes.The original data was qualified after the quality control process,GWAS method was then conducted to analyze the associations.Results: LDSC linkage disequilibrium regression analysis confirmed that the matching of selected cases and healthy controls was reasonable.The capture,typing and GWAS statistical analysis of the target gene regions showed that four SNPs reached the significant level(P<5×10-6).They were gcw171713702 in 2q31.1,rs35093754 in 2q21.3,rs117962550 in 2q37.1 and rs878863933 in 5p15.33.The associated genes were glutamic acid decarboxylase 1(GAD1),lactase(LCT),alkaline phosphatase germ cell(ALPG)and succinate dehydrogenase complex flavin subunit a pseudogene 3(SDHAP3),respectively.Similarity condition analysis showed that gcw171713702 C/T,rs117962550 C/A and rs878863933 T/C were independent susceptibility loci for SSNSWR.There are no reports of gcw171713702,rs117962550 and rs878863933 in the database of expressed quantitative trait loci(e QTL).Conclusion: This is the first GWAS study on SSNSWR,which is the main subgroup of steroid-sensitive nephrotic syndrome.The discovery of GAD1,ALPG,SDHAP3 are located in non-HLA regions that related to SSNSWR that enriched the existing research results of idiopathic nephrotic syndrome(PNS)and further improves the understanding of the pathogenesis of PNS.