| Background and objective:Membranous nephropathy?MN?is a common autoimmune glomerulonephritis characterized by the deposition of immune complexes in the subepithelial space.Although one-third of MN patients achieve spontaneous remission,approximately 40%patients progress to end-stage renal disease?ESRD?in ten years,seriously affecting the quality of life of patients.At present,the diagnosis of MN mainly depends on invasive renal biopsy technique,so it is urgent to seek effective diagnostic markers and specific therapeutic targets on MN.Genomic analyses,which represent major cornerstones of systems biology research,are considered to be unbiased methods for identifying biomarkers and elucidating pathological mechanisms of disease.For example,microarray analysis has been applied to investigate several kidney diseases in humans,such as renal cell carcinoma,diabetic kidney disease,acute kidney injury,and renal graft rejection.In the field of MN,Hauser et.al reported microarray analysis of gene expression in experimental membranous nephropathy and revealed that DNA damage and repair,as well as changes in the extracellular matrix,are major processes in the disease.Nevertheless,the exact pathogenic mechanisms and molecular events underlying MN development remain poorly understood.Thus,the aim of this study was to identify differential gene expression profiles of the MN renal cortex compared to healthy humans and to identify the underlying molecular basis of MN.We performed a whole-genome DNA microarray assay and enrichment analysis to identify relevant biological processes and metabolic pathways regulated by abnormally expressed genes;we also investigated hub genes that may significantly impact those processes or pathways.The data presented herein enhance our knowledge of MN at the genetic level,provide insight into MN pathogenesis,and implicate a new therapeutic strategy for slowing disease progression.Methods:1.Study population.A total of 14 idopathic MN cases were recruited in this study,including seven i MN I–II?Group MN I–II?and seven i MN III?Group MN III?.All the patients were diagnosed and ranked grade I–II or III according to histopathological morphology and immunofluorescence.Punctures of the renal cortex were sampled from each patient before any treatment.Seven healthy controls?Group HC?were recruited.2.Whole-Genome Gene Expression Profiling microarray hybridization.Three puncture samples of the renal cortex from each group were recruited.Total RNA was extracted from each samples using Mini BEST Universal RNA Extraction Kit.Microarray hybridization were performed with total RNA using Illuminar Whole-Genome Gene Expression Directed Hybridization System.Differentially expressed genes?DEGs?between MN I–II vs HC and MN III vs HC were identified.Overlapping differentially expressed genes?ODEGs?were identified in the genes with altered expression in both comparisons.3.Using the Gene Ontology?GO?data to perform BP enrichment analyses and filting hub genes according to the correlation.Using the Kyoto Encyclopedia of Genes and Genomes?KEGG?data to perform pathway enrichment analyses and filting hub genes that shared between KEGG terms.All the hub genes were analyzed by literature search.4.Determination of blood lipid concentration.The fasting venous blood samples were collected from patients in group MN I-II,group MN III and group HC,the level of serum triglyceride?TG?was detected by GPO-PAP method,and the level of serum total cholesterol?TCHOL?was detected by CHOD-PAP method,serum high-density lipoprotein cholesterol?HDLc?and low-density lipoprotein cholesterol?LDLc?were measured by direct method?catalase clearance method?.Using Prism software to analyze the correlation of m RNA levels of hub genes in the renal cortex with circulating TG?TCHOL?HDLc and LDLc concentrations.5.RT-PCR were performed with four total RNA samples in each group using One Step SYBR?Prime Script?RT-PCR Kit II.ACTB was used as internal parameter and 2-??Ct method was used to calculate the relative expression of PLA2G12B.6.Immunofluorescence staining:All renal cortex biopsies of patients with MN?-?,MN III and HC were sectioned,and expression of PLA2G12B protein in MN and HC renal cortex was evaluated by immunofluorescence staining.7.PLA2G12B protein stimulates podocytes in vitro.In order to investigate the effect of PLA2G12B protein on podocytes,human podocytes were cultured in vitro and stimulated with PLA2G12B protein according to time gradient.They were divided into control group?0 h?,0.5 h,1 h,2 h and 6 h groups.The apoptosis of podocytes was detected by flow cytometry,detection of arachidonic acid levels in culture media by high-performance liquid chromatography-mass spectrometry.8.Statistical Analysis.Using SPSS 21.0 software for statistical analysis.Welch's t test was used for inter-group difference analysis.Pearson rank correlation test was used for correlation analysis.P<0.05was considered statistically significant.Results:1.The MN I-II vs.HC comparison detected 291 up-and 659down-regulated genes,meanwhile,in the MN III vs.HC comparison,217 up-and 382 down-regulated genes were detected.Furthermore,167 up-and 291down-regulated genes overlapped between the dysregulated genes in both comparisons.2.Upregulated GO BP enrichment terms,such as triglyceride metabolic process,alanine catabolic process and glyoxylate catabolic process,are related to metabolic processes,whereas the downregulated terms,such as leukocyte activation,leukocyte cell-cell adhesion and leukocyte chemotaxis,are mainly associated with immunologic processes.3.Upregulated ODEGs participate in 16 KEGG pathway,such as cholesterol metabolism,fat digestion and absorption,and the PPAR signaling pathway.Conversely,downregulated ODEGs participate in 13pathways,such as cytokine-cytokine receptor interaction,the IL-17signaling pathway and NF-kappa B signaling pathway.4.38 hub genes were filtered from GO BP and KEGG pathway terms,including the genes PLA2G12B?phospholipase A2 group??,APOA1?apolipoprotein A1?,APOB?apolipoprotein B?,APOC3?apolipoprotein C3?and CETP?cholesteryl ester transfer protein?,which associated with lipid metabolism.5.Several m RNA signal intensities,such as PLA2G12B,CD8A?CD8a molecule?,LTB?lymphotoxin beta?,and NLRP3?NLR family pyrin domain containing 3?were associated with dyslipidemia.6.RT-PCR showed an elevated m RNA levels of PLA2G12B among the MN patients,which were consistent with the microarray result.7.The results of immunofluorescence assay showed that the expression of PLA2G12B was significantly higher in MN I-II group and MN III group than in HC group,but there was no significant difference between MN I-II group and MN III group.8.PLA2G12B stimulated podocytes in vitro.There was no significant difference in apoptosis between 0.5 h group and control group.The percentage of apoptosis in 1h,2h and 6h groups was significantly higher than that in control group,the percentage of apoptotic cells increased gradually.9.The arachidonic acid detection results showed 0.5 h group and 1h group had no significant difference with the control group,but the concentration of arachidonic acid in 2h group and 6h group was significantly higher than the control group.Conclusions:1.The pathways and biological processes related to lipid metabolism,such as cholesterol metabolism and arachidonic acid metabolism play an important role in MN.2.The metabolic pathways related to immune activation,such as IL-17and NF-kappa B signaling pathways are involved in the pathological process of MN.3.PLA2G12B can induce podocyte apoptosis in vitro.4.The podocyte injury in MN maybe induced by PLA2G12B through arachidonic acid metabolism. |
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