Kidney Week

Abstract: FR-PO113

Translational Profiling and RNA-Seq of Proximal Tubule Reveals Gender-Specific Transcriptional Patterns, Tubulointerstitial Signaling, and lncRNA Regulation During Kidney Fibrosis

Session Information

• Molecular Mechanisms of CKD - II
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

• 1903 CKD (Non-Dialysis): Mechanisms

Authors

• Lai, Chun-Fu, Washington University in St. Louis School of Medicine, St. Louis, United States

Chun-Fu Lai,

• Wu, Haojia, Washington University in St. Louis School of Medicine, St. Louis, Missouri, United States

Haojia Wu,

• Chang Panesso, Monica, Washington University in St. Louis School of Medicine, St. Louis, Missouri, United States

Monica Chang Panesso,

• Humphreys, Benjamin D., Washington University in St. Louis School of Medicine, St. Louis, Missouri, United States

Benjamin D. Humphreys,

Background

Proximal tubule epithelial cells (PTEC) are central mediators of interstitial fibrosis in CKD. We applied Translating Ribosome Affinity Purification (TRAP) followed by RNA-seq to precisely measure the gene expression landscape of PTEC during renal fibrosis.

Methods

Bigenic Slc34a1^GFPCreERT2; R26^eGFP-L10a mice received tamoxifen to activate expression of eGFP-L10a in PTEC with subsequent unilateral ureteral obstruction (UUO) surgery. Kidneys were harvested at day 5 or 10. Each group included three female and four male mice. PTEC-specific polysomal mRNA was isolated by affinity purification, and each sample sequenced to a depth of 30 million reads. Extensive informatic analysis identified critical genes and pathways, which were validated by in situ hybridization.

Results

Unexpectedly, we identified a group of genes with sexually dimorphic expression patterns including neuronal regeneration related protein (Nrep), interleukin 34 and nuclear factor interleukin-3-regulated protein (Nfil3). For both sexes, serpin family A member 10 (Serpina10), vascular cell adhesion molecule 1 (Vcam1), SRY-box 9 (Sox9), and nerve growth factor (Ngf) top the list of PTEC-specific highly upregulated genes after UUO. Functional annotations transcriptional changes in PTEC implicate the interleukin-1β and tumor necrosis factor signaling pathways in mediating tubule-interstitial crosstalk. Contrary to published reports, PTEC strongly upregulated Indian hedgehog (Ihh), not Sonic hedgehog (Shh), during renal fibrosis. All results were validated by quantitative PCR and in situ hybridization. Finally, two long intergenic noncoding RNAs (Snhg18 and predicted gene 20513) and microRNA 6358 were strongly upregulated in injured PTEC, suggesting novel regulatory roles in fibrosis. None of these genes have been described in kidney previously.

Conclusion

This is the first comprehensive RNA-seq-based transcriptional profile of PT cells during fibrosis. We reveal unexpected gene expression differences in male vs. female PT, validate novel pathways mediating tubule-interstitial crosstalk and identify novel lncRNA upregulation not previously associated with kidney.

Funding

• NIDDK Support