mRNA Splicing in Podocyte Biology and Glomerular Disease - ABSTRACT / PROJECT SUMMARY Glomerular disease is frequently refractory to current treatments, leading to chronic kidney disease and renal failure, with an annual economic burden of ~100 billion dollars in the US. Podocyte injury, leading to foot process effacement and disruption of slit diaphragm, is a critical determinant of glomerular disease progression. Thus, there is a huge unmet critical need to identify effective podocyte-targeted treatments. We have recently identified crucial glomerular mRNA alternative splicing (AS) events in podocyte injury, demonstrated their functional roles in slit diaphragm and filtration, defined specific trans- and cis-regulators, and devised novel targeted mesoscale nanoparticle delivery methods. These mechanisms provide unexplored opportunities for developing RNA-based diagnostic and therapeutic targets for glomerular disease. The overall objective of this application is to understand the AS events and regulators in podocytes affecting the slit diaphragm and filtration barrier, exploit these mechanisms for therapeutic advantage, and identify diagnostic strategies for SNPs associated with glomerular disease. Our studies using cultured podocytes, organoids, rodent models, and human glomerular disease data have uncovered the glomerular AS landscape, its functional significance in podocyte and slit diaphragm structure and function, and their trans- and cis- regulators, including specific RNA binding proteins and microRNAs (trans), and GWAS-SNPs (cis). The most significant AS candidates, ZO1 (TJP1, tight junction protein 1) and ITM2B (integral membrane protein 2b), are critical SD components, and we found that modulation of their AS variants (α+/α- of ZO1/TJP1, Long/Short of ITM2B) regulated their localization and exerted functional consequences on podocyte viability, adhesion, filopodia formation, architecture of actin cytoskeleton and microtubules, expression of podocyte and slit diaphragm interactors, and barrier function using podocytes, organoid and in vivo models. Furthermore, podocyte injury resulted in dysregulation of miR-184-Splicing factor 1 (SF1) axis and RNA binding protein motif 47 (RBM47), whose knockdown modulated TJP1 and ITM2B AS, podocyte cytoskeleton, viability and markers (nephrin, podocin, podocalyxin, synaptopodin). Our central hypothesis is that mRNA AS of critical determinants of podocyte and slit diaphragm structure and function plays a critical role in glomerular disease, which can be manipulated to develop therapies and utilized as diagnostic tools for podocytopathies. To test this, we propose two Specific Aims: 1) To determine the role of AS of slit diaphragm components in podocytopathies, and 2) To define the trans- and cis- regulation of AS in podocytes. Successful completion of the proposed innovative studies will identify novel mRNA AS mechanisms, that regulate the structural and functional integrity of podocytes, slit diaphragm and glomerular function. Moreover, the project is significant as these mechanisms can be targeted using mesoscale nanoparticle delivery methods with therapeutic splice-switching oligonucleotides, Morpholinos, and anti-miRs. Additionally, this project provides a diagnostic relevance by delving into the functional consequences of SNPs in glomerular disease patients.
