Alternative mRNA Splicing and Polyadenylation Events are Synchronous with Podocyte Injury and Slit Diaphragm Dysregulation in Glomerular Disease Models

Abstract

BackgroundGlomerular disease, characterized by podocyte injury and proteinuria, can lead to chronic kidney disease and end stage kidney disease. We hypothesized that the glomerular pathophysiology is associated with mRNA alternative splicing and polyadenylation of glomerular genes and of critical podocyte and slit diaphragm components that regulate the filtration barrier. MethodsGlomerular damage, accompanied by proteinuria, was induced by puromycin-aminonucleoside or adriamycin to mimic human minimal change disease or focal segmental glomerulosclerosis (FSGS), respectively, and RNA-seq analyses was performed. Alternatively spliced and alternatively polyadenylated events through differential exon and poly(A) site usage were queried in JunctionSeq and APATrap pipelines. These events were further mapped on podocyte and glomerular landscape, analyzed and modulated for slit diaphragm components, and cis- and trans-regulatory elements were identified. ResultsAltered glomerular mRNA processing by alternative splicing/polyadenylation was identified in 136/71 and 1875/746 genes in minimal change disease and FSGS models, respectively. Transcript annotation and prioritization of significant alternative splicing and polyadenylation identified key events in several podocyte and slit diaphragm genes with novel and established roles. Alternative splicing of critical slit diaphragm components, the junction protein TJP1/ZO1 and microtubule associating protein ITM2B was further characterized. Alternative polyadenylation of core members of the slit diaphragm, NPHS1, NPHS2 and NEPH1 was analyzed with potential alteration of microRNA binding sites between the proximal vs distal poly (A) site usage in their mRNAs. Concomitantly, dysregulation of trans-regulatory elements (polyadenylation and splicing factors), was discovered in these models of nephropathies. Additionally, beneficial proteinuria-reducing treatments, pioglitazone and GQ16 reversed many alternatively spliced and polyadenylated events. Moreover, GWAS SNPs as potential cis-regulatory elements were identified in several genes from the human nephrotic syndrome database. Finally, we demonstrated proof-of-concept principle of chemically modified splice switching oligonucleotides in modulating TJP1 alternative splicing in podocytes. ConclusionFindings from our studies identified that glomerular pathophysiology and disruption of the filtration barrier is associated with alternative splicing and alternative polyadenylation of glomerular genes, many of which are crucial determinants of podocyte structure and function and the slit diaphragm complex. Key PointsO_LIAlternative mRNA processing adds an important layer of transcriptome complexity in podocyte and slit diaphragm components during glomerular injury. C_LIO_LITrans- and cis-elements may offer potential mechanisms to regulate alternative splicing and polyadenylation during podocyte injury. C_LIO_LIAntisense oligonucleotides can regulate mRNA processing events, thus offering a potential RNA-based therapeutic strategy to treat glomerular disease. C_LI