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Kidney Week

Abstract: SA-PO778

Characterization of Exosomes Isolated from the Kidneys of Wild-Type (WT) and Tsc1 Knockout (Tsc1 KO) Mice

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Mackenzie, Morgan Elena, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States
  • Zahedi, Kamyar A., New Mexico VA Health Care System, Albuquerque, New Mexico, United States
  • Barone, Sharon L., New Mexico VA Health Care System, Albuquerque, New Mexico, United States
  • Argyropoulos, Christos, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States
  • Soleimani, Manoocher, New Mexico VA Health Care System, Albuquerque, New Mexico, United States
Background

TSC is caused by mutations in TSC1 or TSC2 genes. Cysts are a major renal manifestation of TSC and can lead to end stage kidney disease. In human and mouse models of TSC the cystic epithelium is overwhelmingly comprised of genotypically normal A-intercalated cells (AIC). This suggests the presence of signals that alter the proliferation of AIC. EXO are membrane-bound particles generated by cells and mediate cellular communication, function, and growth. We posit that EXO are important mediators of dysregulated proliferation of AIC lining the cysts in TSC. To this end, we isolated, characterized, and identified the differences in the RNA content of EXO from WT and Tsc1 KO mice.

Methods

EXO from kidney explants were isolated by size exclusion chromatography. The isolated EXO were characterized by western blot analysis, transmission electron microscopy (TEM), and fluorescent nanoparticle tracking analysis (fNTA). EXO RNA was isolated and run through cDNA library preparation methodology for Nanopore sequencing and mapped to the mouse transcriptome.

Results

TEM studies showed that EXO sizes range from 50 to 300nm. The fNTA studies revealed that greater than 70-77% of isolated particles were EXO with a size range of 50 to 300nm. The EXO were of similar size and distribution; however, the number of EXO was significantly higher in the preparations from the kidneys of Tsc1 KO mice. Isolated EXO were highly enriched for CD63 and RAB27A markers. RNAseq analysis revealed that 145 transcripts were differentially represented (p<0.05) in the EXO isolated from the kidneys of Tsc1KO vs. WT mice. These included 24 lncRNAs (4 previously identified), 1 vtRNA, 107 mRNAs, and 8 pseudogenes. Enrichment analysis revealed numerous pathways that differed between Tsc1 KO and WT exosomes, with significant fold enrichment found in the mTOR and phospholipase D signaling pathway and those of glycerophospholipid metabolism. The mRNA for SLC25A26, a mitochondrial protein that functions in S-adenosylmethionine (SAM) S-adenosylhomocysteine (SAH) exchange, and Lpin2 were substantially enriched in Tsc1 KO, mice.

Conclusion

LPIN2 and SLC25A26 may affect mTOR activation via SAH levels and DAG signaling, respectively, pointing to a potentially novel mechanism in TSC cystogenesis.

Funding

  • Other NIH Support