| Background:Lupus nephritis is a manifestation of System Lupus Srythematosus?SLE?implicated kidney.It is also the main cause of death for the patients.It has long been considered as a dangerous and poor prognosis glomerulonephritis.Lupus nephritis is the most common complication of systemic lupus erythematosus.Almost all patients with systemic lupus erythematosus suffer from histological,immunological and ultrastructural changes of renal tissue.Statistical data from the Institute of Nephrology of PLA Nanjing General Hospital show that lupus nephritis reached 13.5%of the total kidney disease,while 54.3%of secondary glomerulonephritis[1].Systemic lupus erythematosus?SLE?is a complex autoimmune disease characterized by a variety of autoantibodies and diversity.30%-50%patients with systemic lupus erythematosus get clinical manifestation of renal damage in the early stages of the disease,as no symptoms of abnormal urine,nephrotic syndrome,acute nephritis syndrome,rapidly progressive glomerulonephritis?RPGN?,whichisthe most serious.The clinical and immunological features of 1352 cases of lupus nephritis in the Institute of Nephrology of PLA Nanjing General Hospital,with a total of 17.5%acute renal failure;while the foreign clinical data suggest that the performance of rapidly progressive glomerulonephritis reached 30%.Acute renal failure complicated with lupus nephritis associated with the following factors:the diffuse crescent formation in glomerular;thrombosis formation in the glomerular capillary loop;the lupus renal vascular lesions,such as thrombotic microangiopathy?TMA?;the acute interstitial nephritis;the renal vein thrombosis,occasionally renal artery thrombosis.If the active lesion is not effectively controlled,the patient couldget into chronic renal failure.8%-15%of the lupus nephritis patientseventually fall into endstage renal disease?ESRD?,the most in the patients who suffered type IV,type IV and V lupus nephritis.Lupus nephritis has a variety of pathological types.The IV type is diffuse lupus nephritis,more than 50%glomerular involved.The lesion can be expressed as active or inactive,segmental or glomerular capillaries,capillaries or extra capillaries proliferation.The pathogenesis of lupus nephritis related to genetic background and environmental factors,sex hormone metabolism,reduce the number of Treg,the abnormal proliferation of B cell activation,antibody generation,abnormal complement activation and immune complex deposition in kidney and other factors,there are very complex regulatory networks.The IgG subtypes of different types of lupus nephritis are different.In prol iferative lupus nephritis,IgG is mainly IgG1 and IgG3,activating complement through classical and alternative pathways[2].In type V lupus nephritis,IgG is mainly IgG4,which mainly activates the complement system by alternative pathway[3].Nevertheless,the pathogenesis of lupus nephritis is still not fully elucidated.The KDIGO guidelines require a renal biopsy for lupus nephritis to determine a treatment based on the pathological features of the kidney[4].In particular,when cellular crescents are detected in renal biopsy,it means that rapid renal failure is occurring.In this case,if a large dose of glucocorticoid or cyclophosphamide is not given timely treatment,the patient's renal failure will be difficult to recover.The formation of glomerular crescent crescent,which has serious adverse effects on the prognosis of nephritis,has attracted more and more scholars'attention in recent years.For this reason,the 2017 Oxford Classification of IgAN added MEST-C scoring for cellular/fibrous crescents?a MEST-C score?C0:no crescent;C1:0-25%cresent;C2:?25%cresent?to assess the significant impact of the presence of crescents on the kidneys[2].In the case of crescent nephritis,the KDIGO guide also recommends the use of steroids and cyclophosphamide.Once the treatment window is missed,the patient will suffer gradually deteriorating renal failure.And if the type IV lupus nephritis is combined with a cell crescent body,a more rapid progressive renal dysfunction will occur.How to detect progressive renal damage at early stage in lupus is a great challenge for clinicians.Albuminuria is usually used as a biomarker for kidney damage.However,it does not have a complete correlation with the pathological features of the kidney.However,due to the invasiveness of kidney biopsy,biopsy cannot be performed in patients with lupus nephritis frequently.We need to find reliable biomarkers for non-invasive diagnosis and long-term monitoring.MiRNA is a small non coding RNA,which mainly regulates cell differentiation,growth,apoptosis and proliferation by regulating the expression of posttranscriptional genes,degrading mRNA or inhibiting transcription.MiRNAs plays a key role in the normal growth and development of kidney and function mediation.In recent years,its role in renal homeostasis and disease has also attracted the interest of researchers[5,6].MiRNA is released from the cell into the urine and can bind to the RNA binding protein or pack into the exosome[7–9].With the promotion of body fluid biopsies,more and more attention has been paid to the detection of exosomal miRNA as a biomarker for various diseases.The exosome is a small vesicle of 30-120nm in diameter and exists in almost all biological fluids,including blood,urine,saliva,milk,cerebrospinal fluid,etc[10–13].Exosome,as a subtype of extracellular vesicles,differs from other vesicles in that they have different biological origins in cells:mature multivesicular bodies fuse cell membranes to release exosomes to extracellular space.Exosome contains miRNA,lncRNA,mRNA and proteinetc[14,15],which transfer biological information from origin cells to target cells.There are clear evidence that all cells of nephrons can excrete exosomes[15][16].Exosomes can accurately represent renal dysfunction and structural damage,which is considered an important way to disseminate information[17].Therefore,urinary exosomes can be used as potential biomarkers for diagnosis and prediction of prognosis[18,19].Some urine miRNAs come from miRNAs filtrated from the circulation,but more of them are from nephron-derived exosome miRNAs[20,21].Recent studies have shown that the expression profiles of urinary exosome miRNAs have different characteristics in patients between FSGS and MCD[22],type I and type II diabetic nephropathy[23,24].The level of miR-29c expression in urinary exosomes can predict early fibrosis in lupus nephritis[25].This suggests that:the urinary exosomes miRNA expression profiles accurately reflects the pathophysiological characteristics of the kidney,opening a new window for continuous monitoring of renal pathology to the study of renal pathological mechanism and early diagnosis and treatmen.In the present study,we first investigated the expression profile of type IV lupus nephritis with crescents by sequencing urinary exosome miRNA,and selected differentially expressed miRNAs for RT-qPCR validation to screen out non-invasive diagnostic markers for the disease.Objective:In this study,we from morning urinary exosome of the activity of type IV lupus nephritis with cellular crescents,the activity of type IV lupus nephritis without cellular crescents,inactive IV lupus nephritis and healthy people to explore the expression profile of miRNAs in each group and analysis the expression characteristics and regulation of the network,looking for noval biomarkers for type IV lupus nephritis and cellular crescents.Methods:1.Sample source and groupingAll lupus nephritis patients were admitted to the nephrology department of the First Affiliated Hospital of Army Medical Universityfrom 2016 to 2018.All patients received renal biopsy,and the pathological classification was confirmed,and the presence of cell crescent was determined.Healthy volunteers were derived from the medical examination center of the hospital as a control group in the study.Healthy people are defined as no systemic disease such as lupus,hepatitis B,diabetes,hypertension,renal function or eGFR normal,no hematuria,proteinuria or leukocyte.The Helsinki declaration,revised in 2008,was approved by the ethics committee of the First Affiliated Hospital of Lu Military Medical College?Southwest Hospital?.After signing the informed consent,a database of screening groups is set up:We collected morning urine 100ml from each active type IV lupus nephritis with cell crescent,activetypeIV lupus nephritis without cell crescent,inactive type IV lupus nephritis and healthy people,and processed it in 1 hour;2.Extraction of urine exosomeExtraction of exosome from urine by gradient centrifugation:Initially centrifugation2500g,30min to remove cells and residue,at 4 centigrade 17000g centrifugation 30min removal of large vesicles;the ultracentrifuge centrifuged 200000g for 70min at 4?and removed the supernatant to obtain the preliminarily granulation of the exosome;ultracentrifuge centrifuged 200000g for 70min at 4?again to get the pallet exosomes,add PBS to-80 C ultra low temperature refrigerator for lateruse;3.Transmission electron microscopy testTransmission electron microscopy was used to detect the morphology and diameter of the exosome,and the appearance of the exocrine was confirmed.We placed a drop,approximately 30?L of exosomes resuspended in PBS on the paraphilm.Aformvar carbon-coated Copper grid was positioned on top of each drop for 10 minutes.Finally,a drop of 1%uranyl acetate was placed on the paraphilm and incubated the grid on top of the drop for 10 minutes.After air drying,the grids were examined with aelectronic transmission electron microscope made in Japan?JEM-1400/JEM-1400 PLUS,Japan?.The size distribution of captured exosomes in a total of 4 micrographs from two samples was analysed using an image processing program;4.ZETASIZER Nano series-Nano-ZS testThe size distribution of exocrine was detected by Nano series-Nano-ZS,and the size distribution of exocrine was confirmed.Add PBS to suspendexosome.Select disposable clean sample pool,wipe with dustless paper,ensure the light path,cover the outer tube wall in the morning,slowly inject the external body solution and avoid bubbles.Seal the sample pool with a lid and put it in the instrument.Samples were examined using ZETASIZER Nano series-Nano-ZS,according to manufacturer's instructions.5.Detection of surface markers by flow cytometryTake out the exosome of the refrigerator at-80?and add the PBS tosuspension.Two samples?number DCX,HSF?were used as negative control and labeled as NC.The other parts were stained with CD63 and CD81 two groups,labeled CD63 and CD81,respectively.Samples were examined using BD accuri C6 flow cytomenter,according to manufacturer's instructions.6.Western blotIdentify the exosomal markers of CD9,CD81 and the non exosome surface marker Calnexin weatherexist by Western blot.The exosomes of urine were lysed in RIPA lysis buffer on ice,and then centrifuged at 12,000 g for 15min.The supernatant was collected and transferredto a new EPtube.An equal amountof total soluble protein were electrophoresed on 8%SDS-PAGE?Life Technologies,USA?and transferred to a PVDF membrane for each sampleanalysed.Themembranes was incubated with primary antibodies CD9 rabbit dilution1:1000 and CD81 rabbit dilution1:1000,while non-exosomal primary antibodies:calnexinmousepolyclonaldilution 1:1000?Abcam,UK?and appropriate horseradishperoxidase-conjugatedsecondaryantibodies.Blotswere visualizedwithchemiluminescence reagents?Beyotime,Shanghai,China?.7.Illumina HiSeqTM 2500 sequencingThe miRNA expression profiles of each group were obtained by Illumina HiSeqTM2500 sequencing,and the statistical difference was calculated for the deregulatedmiRNAs.Exosomes were isolated and characterized as described above.Total RNA from exosomes was used for miRNA sequencing.Library preparationand miRNA sequencing were performed by Ribobio?Guangzhou,China?.Briefly,total RNAsamples were fractionated and only small RNAs nts were used for librarypreparation.After amplification by PCR,products were sequenced using the Illumina HiSeq2500 platform.8.Heatmap HCLUsing Multi Experiment Viewer?V4.9.0?software to choose Gene Tree and Sample Tree to do HCL analysis with Optimize Gene Leaf Order and Optimize Sample Leaf Order setting to get Heatmap between type IV lupus nephritis and healthy people,Heatmap between active IV type lupus nephritis and inactive IV type lupus nephritis,Heatmap between active IV type lupus nephritis and healthy people,Heatmap between active IV type lupus nephritis with cellular crescent and active IV type lupus nephritis without cell Crescent.9.Volcano plot analysisThe sequence data of these groups were input to E Chart,and the Volcano plot atlas was analyzed and to obtain expression profile and|Fc|level of deregulatedurinary exosomal miRNAsbetween type IV lupus nephritis and healthy people,active IV type lupus nephritis and inactive IV type lupus nephritis,active IV type lupus nephritis and healthy people,active IV type lupus nephritis with cellular crescent and active IV type lupus nephritis without cell Crescent.10.Path analysis of deregulated miRNAsUsing DIANA miRPath?V3.0?to analyze the pathway analysis of significant differences between the above groups of miRNAs to explore the possible signal pathways involved in the differential expression of miRNAs under different pathological conditions.Intersection of the DIANA-mirPath results of 3 databases?TargetScan,Tarbase7.0,microT-CDS?V5.0??were get,after excluding tumor,hepatitis B,leukemia and thyroid hormone related pathways,we got the following pathways,P value and gene and miRNAs related to IV LN and cell crescentic;11.TF predictionTF?transcription factor?prediction using the PSSM?Position-Specific Scoring Matrices?matrix of each model in the JASPAR database;The TESS software developed by the computational biology and Informatics Laboratory of University of Pennsylvania was applied to search for the binding site of TF based on the weight matrix database according to the imbalance of TF binding sites in the genome;Using the FIMO software package to use the MEME?Multiple EM for Motif Elicitation?matrix in the search sequence of the known motif to search the matching loci of the potential motif in the sequence.The prediction results of TF obtained by the three prediction software are synthesized and the TF can be predicted by the three methods as candidate TF.12.miRNA target gene predictionUsing TargetScan,miRDB,miRTarBase and miRWalk the four softwares to predict target gene of deregulated miRNAs between type IV lupus nephritis and healthy people,active IV type lupus nephritis and inactive IV type lupus nephritis,active IV type lupus nephritis and healthy people,active IV type lupus nephritis with cellular crescent and active IV type lupus nephritis without cell Crescent.The results of miRNA target genes obtained from the four prediction software were intersected,and the results predicted by various algorithms and experimental results were collected as candidate genes for miRNAs differences among groups.13.TF-miRNAs-mRNA networkThe candidate TF,miRNAs and miRNAs,in accordance with the combination of regulation betweenTF,miRNAand miRNA gene targete,is established with Cytoscape network visualization to analysis interrelation of them.14.RT-qPCR verificationFilter the miRNAs and set up a validation group.Morning urine of active type IV lupus nephritis with cellular crescentic,active type IV lupus nephritis without cellular crescentic,inactive type IV lupus nephritis and healthy people were collected to extract exosomal miRNA to do RT-qPCR to verify expression level.3 miRNAs?miR-3135b,miR-654-5p,miR-146a-5p?were selected to do the test to identify the result from sequencing was accurate and reliable.15.ROC analysisUsing SPSS?V 19.0.0?to generate ROC of miR-3135b,miR-654-5p,miR-146a-5p,analysis AUC,Youden Index and p valve.The sensitivity and specificity of these 3 miRNA in the diagnosis of IV type lupus nephritis with cell crescent body were calculated respectively.Conclusions:1.By high-throughput sequencing,there were differential expression of miRNAs in active type IV lupus nephritis with cellular crescentic,active type IV lupus nephritis without cellular crescentic,inactive type IV lupus nephritis and healthy people were collected.2.Analysis of the differential expression of miRNAs in the groups obtained 15 signal pathways associated with cell crescent in type IV lupus nephritis.3.Based on bioinformatics analysis of multiple databases,we predict candidate TF and mRNA,to build a coexpression network based on miRNA,including interaction relationship between TF,miRNA and mRNA.4.3 miRNAs?miR-3135b,miR-654-5p,miR-146a-5p?were selected to do RT-qPCR and the ROC curve was plotted.It was found that miR-3135b and miR-654-5p could distinguish active type IV lupus nephritis with or without celluar crescent.5.In conclusion:these results indicate that the formation of celluar crescent in type IV lupus nephritis involves complex signaling pathways and regulatory networks.Urinary exosomal miR-3135b and miR-654-5p can be used as biomarkers for diagnosis of IV type lupus nephritis with cell crescentic. |
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