Proximal Tubule Alternative Splicing and Progression of Diabetic Kidney Disease

Washington University, Saint Louis MO

Investigators

Abstract

ABSTRACT Diabetes mellitus affects ~10% of the U.S. population and diabetic kidney disease (DKD) is the leading cause of end stage kidney disease (ESKD) worldwide. Despite recent treatment advances, only a limited reduction in rates of ESKD from DKD have been observed. While sodium-glucose cotransporter 2 inhibitors (SGLT2i) have emerged as a promising drug class for the treatment of DKD, their mechanism of action remains unknown. Understanding the renoprotective effects of SGLT2i will inform the development of future therapeutics. Recent work from our laboratory generated a single nucleus RNA-sequencing (snRNA-seq) atlas of a murine model of DKD and response to five different treatment regimens, including SGLT2i. This ~1 million cell dataset revealed that diabetes downregulated the spliceosome regulator Srsf7 in PT and its expression was specifically rescued by SGLT2i - but not other therapeutic regimens. The spliceosome regulates alternative splicing, a process that increases transcript diversity by generating multiple variant mRNAs from one gene. We further found that DKD drove changes in alternative splicing in vivo, and in vitro, PT knockdown of Srsf7 triggered alternative splicing of ~250 genes and differential gene expression inducing a pro-inflammatory phenotype. There is surprisingly little known regarding alternative splicing in both kidney health and disease. Based on our preliminary data, we propose that DKD induces tubulointerstitial fibrosis by downregulation of the spliceosome gene Srsf7 in the S1 segment of the PT, leading to a pro-inflammatory and pro-fibrotic transcriptome. We further hypothesize that alternative splicing is a broader PT response to chronic injuries that is conserved in humans. We propose the following investigations to critically test the importance of alternative splicing in regulating the proximal tubule injury response to the diabetic milieu and chronic kidney disease: Specific Aim 1: Define and validate the spectrum of alternative splicing in DKD and treatment with SGLT2i using long-read RNA sequencing. Long read sequencing is critical for accurate quantitation of alternatively spliced isoforms and has never been performed in DKD models. Specific Aim 2: Test the hypothesis that hypoxia drives PT alternative splicing via downregulation of SRSF7. Our preliminary data indicates that PT cells exposed to hypoxia or a hypoxia mimetic downregulate SRSF7, and cells with SRSF7-knockdown have decreased cell proliferation and upregulation of inflammatory pathways. Specific Aim 3: Determine whether regulation of alternative splicing occurs in human DKD and whether Srsf7 is required in the proximal tubule to promote a `healthy' transcriptome. Whether or not alternative splicing in kidney occurs in human DKD is unknown, and whether Srsf7 is required for PT homeostasis is undefined.