Abstract: TH-PO245

Mapping the Spatiotemporal Transcriptional Landscape of the Collecting Duct During Ischemic Injury Using Non-Invasive Optical Guidance

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Paragas, Neal A., University of Washington, Seattle, Washington, United States
  • Miyazaki, Tomoaki, University of Washington, Seattle, Washington, United States
  • Hsu, Yun-Wei A., University of Washington, Seattle, Washington, United States
  • Gharib, Sina, University of Washington, Seattle, Washington, United States
Background

Collecting duct (CD) cells are sensitive to acute kidney injury (AKI) before an appreciable rise in serum creatinine. CD cells are mitochondrial rich and sensitive to oxidative stress (OS), so we specifically targeted them to monitor H202 generation along with changes in gene expression.

Methods

To interrogate genetic changes at the peak of OS, we non-invasively monitored H202 generation to identify the most relevant time points tissue acquisition for CD specific RNA-seq. We created a CD, HoxB7-luciferase (CD-Luc) bioluminescent reporter animal, to monitor OS. CD-Luc cell-specific reporter mice were injured by 30 min bilateral ischemia reperfusion injury (IRI) and H202 activity monitored non-invasively. A H202 sensitive caged-luciferin substrate was used to quantify cell specific changes of H202 after IRI. In parallel, we created a CD, HoxB7-riboTRAP (CD-riboTRAP) RNA isolation animal, to selectively immuno-precipitate (ip) RNA transcripts from CD cells. CD population was RNA sequenced and analyzed.

Results

In vivo measures of CD-Luc H202 activity were in agreement with MDA levels (lipid peroxidation), and qPCR of biomarkers of hypoxic stress (Hspa1b and Hmox1) with all peaking at 6-9 h after IRI. In the parallel, CD-riboTRAP IRI animals 9 h after injury had ip RNA-seq. We identified over 14,000 transcripts with many up- and down-regulated genes between IRI vs. sham CD-riboTRAP populations. Previously validated biomarkers of renal injury to the medulla were upregulated (Lcn2, Clu, and Spp1). Pathways involved in mitochondrion-associated processes were down-regulated. We pretreated CD-Luc before IRI with a targeted mitochondrial antioxidant and were able to reverse the CD OS in vivo.

Conclusion

In sum, using our novel integrative approach we: (i) non-invasively and longitudinally imaged the peak time point of OS in the CD; (ii) used ip to isolate CD RNAs at that time point; (iii) successful carried out RNA sequencing; (iv) performed differential gene expression and pathway analysis to reveal mitochondrial OS as a potential target pathway; (v) reversed the acute OS state with a targeted mitochondrial antioxidant with reduced lipid peroxidation in the kidney; and (vi) validated the reversal of mtROS induction in ROS reporter animal model and as a result suppressed the strong inflammatory stimulus of IRI.

Funding

  • NIDDK Support