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Abstract: TH-PO751

Nephrin, Podocin, and Neph1 Encoding mRNAs Are Dysregulated by Alternative Polyadenylation During Podocyte Injury

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Das, Monoj Kumar, Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
  • Webb, Amy, The Ohio State University, Columbus, Ohio, United States
  • Bryant, Claire, Forge Biologics, Columbus, Ohio, United States
  • Moore, Claire L., Tufts University, Medford, Massachusetts, United States
  • Agrawal, Shipra, Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States
Background

Podocyte injury and proteinuria are hallmark characteristics of glomerular disease. Nephrin, Podocin and Neph1 are critical regulators of the structure and function of podocytes and the glomerular filtration barrier. We hypothesized that these important components of the slit diaphragm could be dysregulated at the level of mRNA processing during podocyte injury and glomerular disease.

Methods

Glomerular alternative mRNA polyadenylation was detected using APATrap analyses of the RNASeq data from puromycin aminonucleoside (PAN) and adriamycin (ADR)-induced nephropathy models of podocyte injury and glomerular disease, mimicking human minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), respectively.

Results

Nphs1 (encoding for Nephrin) mRNA presented with a significant shift from proximal to distal usage of the polyadenylation [p(A)] site in its 3’ untranslated region (3’ UTR) in both PAN (12.7% shift vs healthy control) and ADR (24% shift vs. healthy control) -induced glomerular disease models. The Nphs1 mRNA p(A) shift to distal site and lengthening of the 3’UTR during injury was also validated by 3’ Rapid Amplification of cDNA Ends assay. In contrast, a reverse shift was detected on the Nphs2 (encoding for Podocin) mRNA in the ADR-induced nephropathy model (9.2% shift vs healthy control) only. The p(A) shift in Nphs1 mRNA to a more distal site during injury results in introduction of target sites for miRNAs (miR-376a-5p, miR-466c-5p and miR-466d), which might affect its localization, stability and translation. Neph1 mRNA also showed a shift to a more distal site (~13% shift vs healthy control) in both PAN and ADR -induced glomerular disease models, exposing additional 415 nucleotides to miRNA targeting. On the other hand, a shift in Nphs2 p(A) site from distal to proximal during injury results in introduction of the p(A) site in its last coding exon upstream of the STOP codon, which might affect its stability by the mRNA non-STOP decay pathway.

Conclusion

Alternative mRNA polyadenylation of Nphs1, Nphs2 and Kirrel mRNAs during podocyte and glomerular injury could result in their altered levels or function, with potential implications towards the dysregulation of podocyte structure and function and disruption of glomerular filtration barrier.

Funding

  • Private Foundation Support