Investigating The Pathogenesis Of IgA Nephrolopathy By Single-cell Transcriptome Sequencing

Backgroud:Immunoglobulin A nephropathy(IgAN)is the most common primary glomerular disease worldwide currently.It is particularly common in China and one of the common causes of end stage renal disease(ESRD).Due to the diverse clinical manifestations and the complex pathological features,IgAN patients have different response to therapy,and the optimal treatment for IgAN remains controversial.Early diagnosis and intervention can improve the prognosis and reduce the incidence of ESRD.At present,the pathogenesis of IgAN is still unclear.IgAN is considered to be an autoimmune disease.Multiple-hit pathogenesis is now widely accepted.Based on the hypothesis,B lymphocytes is the crucial role,because IgA1 with galactose-deficient Olinked glycans in the hinge region,secreted by B lymphocytes,increases significantly in IgAN patients.Circulating IgA1 is derived from peripheral blood B cells,however there is a lack of in-depth research on the classification of peripheral blood cells in patients with IgAN.In recent years,the development of single-cell sequencing technology has solved the problem of cell heterogeneity.Cells can be clustered and annotated by unsupervised clustering,which is helpful for the study of specific cell types.Therefore,our study investigated the pathogenesis of IgAN by single-cell transcriptome sequencing,specifically focusing on B lymphocytes.Methods:Single-cell transcriptome sequencing was performed on peripheral blood monocytes derived from 3 IgAN patients and 2 healthy controls,and differential gene expression profiles of peripheral blood single-cell were established.Functional analysis was performed to explore the pathogenesis of IgAN.Meanwhile,RT-PCR was used to validate the differential expression of mRNA and miRNA,which verified the reliability of single-cell transcriptome sequencing results.To explore the association of GWAS loci about intestinal inflammation and IgAN,we used single-nucleotide polymorphism analysis,immunohistochemistry and multiple immunofluorescence staining.EBV-miRNA-BART19-3p,differentially expressed in expression profile of microRNAs from peripheral blood B lymphocytes in IgAN patients,was verified by RTPCR.Gd-IgA1 level in serum was detected by ELISA.Transfection of EBV-miRNABART19-3p mimic to DAKIKI cells was used to explore the effect on IgA1 O-glycosylation of IgAN.In addition,detection of EBV antibody levels in serum and EBV DNA expression levels in plasma to explore the pathogenic mechanism of EBV infection in IgAN patients.The levels of α1-antitrypsin in serum,urine and kiney tissue were detected by ELISA,immunohistochemistry and multiple immunofluorescence staining.Stimulating DAKIKI cells with lipopolysaccharide(LPS)was used to establish a non-specific inflammatory cell model and explore the effection of inflammation on SERPINA1.Results:We successfully established the differential gene expression profile of peripheral blood single cells in IgAN.RT-PCR of B lymphocytes demonstrated that SPI1,MXD1 and S100A9 mRNA levels were higher in IgAN patients than controls,which showed the reults of single-cell transcriptome sequencing was avalible.Functional analysis revealed that differential genes were extensively enriched in inflammation / infection-related pathways in each cell type,and the EBV infection pathway focused on antigen presentation.In the inflammation model of B lymphocyte,the expression levels of SPI1,MXD1 and S100A9 of DAKIKI cell increased after LPS stimulation.According to analyse GWAS loci of IgAN,we found that CARD9,VAV3,PSMB8 and PSMB9 were IgAN susceptibility genes related to intestinal inflammation.The interaction of rs4077515 and rs17019603 could increase the susceptibility to IgAN.For additive interaction,the CT or TT of rs4077515 and GG of rs17019602 genotype combination conferred a 2.56-fold risk of IgAN reference to CC of rs4077515 and AA of rs17019602(OR=2.56,95%CI: 0.98–6.69,P = 0.049).IgAN peripheral blood single cell transcriptome sequencing revealed that PSMB8 and PSMB9 expression levels were elevated.Mouse kidney single cell sequencing and immunohistochemistry and fluorescent staining of human kidney tissues proved that PSMB8 and PMB9 were localized in the second novel cell.EBV-miRNA-BART19-3p was highly expressed in IgAN B lymphocytes(P = 0.024),but it may not influence the glycosylation of IgA1.Increased EBV-miRNABART19-3p can lead to highly expressed MXD1 mRNA.However,detection of the expression levels of EBV antibodies in the serum of IgAN patients and controls revealed that the previous infection rates were above 80%,and there was no significant difference in the EBV infection between two groups.The positive rate of EBV DNA in plasma was low and there was no significant difference between IgAN patients and controls.Glycoprotein profiling analysis in urine showed that α1-antitrypsin level significantly increased in IgAN patients,and its coding gene,SERPINA1,was significantly up-regulated in IgAN peripheral blood single cell sequencing.Validation experiments showed that SERPINA1 mRNA level of B lymphocytes was much higher in IgAN patients than controls(8.75 ± 6.23 vs.3.02 ± 1.80,P = 0.007).In addition,α1-antitrypsin was also increased in kidney tissue,especially in capillary loop and mesangial area,but α1-antitrypsin in serum didn’t increase significantly IgAN patients.In clinical analysis,it was found that urine α1-antitrypsin was negatively correlated with serum GdIgA1 expression level,and SERPINA1 mRNA expression level in B lymphocytes was positively correlated with serum C3 and C4.The result above explained that respiratory tract infections were related to IgAN.Conclusion:Differential gene expression profiles of IgAN in peripheral blood single cells were successfully established,and it demonstrated that the inflammation/infection pathway was associated with IgAN.The results derived from the three branches of intestinal infection,respiratory infection and non-specific inflammation,further proved that inflammation was tightly related to IgAN.Increased expression of α1-antitrypsin in IgAN was proved from multi-angles,and its clinical significance needed further exploration.