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Abstract: PO0626

Transcriptomic Profiling Identifies Potential Mediators of Tubular Injury Sensitization of Glomeruli to Subsequent Second Hits

Session Information

  • CKD Mechanisms - 2
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Babickova, Janka, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Li, Xin, Shanghai Jiao Tong University, Shanghai, China
  • Yang, Haichun, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Fogo, Agnes B., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background

Previous studies have shown that even isolated mild tubular injury leads to more severe glomerular damage in response to subsequent injury. The responsible mediators for this sensitization are unknown.

Methods

Double transgenic male mice, Nep25/DTR+ (expressing human CD25 receptor on podocytes and Diphtheria toxin receptor on proximal tubular cells) and Nep25/DTR- (n=5 per group) were used. Tubular injury was induced by injecting diphtheria toxin, followed by uninephrectomy (Nx) 4 weeks later and induction of glomerular injury by LMB2 toxin (CD25 ligand) one week after Nx. Mice were sacrificed 4 weeks after LMB2. Glomeruli and tubules from Nx were separated by sieving technique, RNA was isolated from tubules and next generation RNA sequencing was performed.

Results

Histopathological analysis and urinary Kim-1 at Nx and sacrifice confirmed mild tubulointerstitial fibrosis at Nx in Nep25/DTR+ but not Nep25/DTR- mice, and more severe glomerulosclerosis and albuminuria at sacrifice after LMB2 in Nep25/DTR+ vs Nep25/DTR- mice. RNA sequencing revealed 283 differentially expressed genes between the groups, with 93 over-represented and 190 under-represented in Nep25/DTR+ vs Nep25/DTR-. GO of biological processes showed involvement in 13 processes, with the highest amount of genes involved in cellular processes, biological regulation and metabolic processes. STRING analysis of protein-protein interactions (PPI) based on cellular processes detected interactions between the Serpin family members: plasminogen activator inhibitor PAI-1 (Serpine1), alpha-1-antitrypsin 1-2 (Serpina1b), protein Z-dependent protease inhibitor (Serpina10) and complement C4b (C4b). In addition, members of the non-canonical Wnt signalling pathway Wnt-9a (Wnt9a) and Wnt-10a (Wnt10a) and their interactors latent transforming growth factor beta binding protein 2 (Ltbp2) and VANGL planar cell polarity protein 2 (Vangl2) were over-represented and PPI of these genes was found. Quantitative real-time PCR confirmed numerically higher expression of all above-mentioned genes in Nep25/DTR+.

Conclusion

High-throughput RNA sequencing of isolated tubules with mild injury revealed potential novel mediators of glomerular sensitization to a subsequent injury. Further experimental validation of the effects of the identified molecules on glomerular injury are warranted.

Funding

  • NIDDK Support