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Abstract: SA-PO429

Long-Read Sequencing Identifies Loss of Proximal Tubule Spliceosome Gene Srsf7 as a Driver of Inflammation Through Alternative Splicing

Session Information

Category: Diabetic Kidney Disease

  • 701 Diabetic Kidney Disease: Basic

Authors

  • Noonan, Megan L., Washington University in St Louis School of Medicine, St Louis, Missouri, United States
  • Wu, Haojia, Washington University in St Louis School of Medicine, St Louis, Missouri, United States
  • Humphreys, Benjamin D., Washington University in St Louis School of Medicine, St Louis, Missouri, United States
Background

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide, and SGLT2i have potent reno-protective effects, however the mechanism is unclear. Recently, the mRNA splicing factor Srsf7 was shown in mice to be downregulated in DKD, and SGLT2i treatment specifically rescued Srsf7 expression in the S1-segment of the proximal tubule (PT).

Methods

hTERT-RPTECs were treated with control or siRNA against SRSF7 for 72h, then submitted for long-read (Nanopore) sequencing to more accurately capture alternative splicing. Reads were aligned using minimap2 then quantified with Salmon and analyzed for differential gene expression and differential transcript usage (DTU) using the R packages IsoformSwitchAnalyzeR and bambu.

Results

SRSF7-knockdown (KD) efficiency was >80%. Differential gene expression analysis also identified SRSF7 as a top significantly down-regulated gene. RSAD2 and OAS2, genes involved in viral inflammatory response, were identified as top upregulated genes, which was confirmed by significant increases in both gene and protein expression in SRSF7-KD cells. Gene Ontology analysis showed upregulation of biological processes related to viral response, NFkB signaling, and response to interferon, well as enrichment of these hallmark inflammatory pathways by GSEA. Conversely, processes related to cell cycle were downregulated in cells with SRSF7-KD, suggesting changes in proliferation. MKI67 gene expression and BrdU incorporation were decreased in SRSF7-KD cells. The genes SNAPIN, LGMN, and GGCT were identified by both computational tools as having alternatively spliced isoforms between control and SRSF7-KD cells and were validated using RT-PCR. Genes with alternative splicing were enriched in pathways related to viral response/inflammatory signaling, mRNA alternative splicing, RNA metabolism, and apoptosis.

Conclusion

Decreased SRSF7 in PT cells drives expression of pro-inflammatory genes and regulates splicing of inflammatory genes. Pro-inflammatory alternative splicing events in the S1 segment may thus drive DKD progression and SGLT2i are reno-protective by promoting an anti-inflammatory S1 transcriptome by rescuing Srsf7 expression.

Funding

  • NIDDK Support