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Kidney Week

Abstract: PO0216

Characterizing De Novo Lymphangiogenesis During AKI Using 3D Imaging and Tissue Cytometry

Session Information

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

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Black, Laurence Marie, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Winfree, Seth, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Kamocka, Malgorzata, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Khochare, Suraj Deepak, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Traylor, Amie, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Esman, Stephanie, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Khan, Shehnaz, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Zarjou, Abolfazl, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Agarwal, Anupam, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

The renal lymphatic system is essential for fluid and electrolyte homeostasis, lipid and cholesterol transport, and immune surveillance with lymphatic vessels (LV) primarily intertwined with the blood vasculature. LV development, or lymphangiogenesis (LA) is regulated by its master transcription factor, prospero-related homeobox-1 (Prox-1), which determines lymphatic cell fate. LA is accentuated during inflammation or injury states such as acute kidney injury (AKI), though mechanisms of LA in AKI remain unclear. Understanding the LA process is essential because it will elucidate potential therapeutic targets in AKI.

Methods

Using 10-week old male Prox1-tdTomato lymphatic reporter mice (ProxTom), we investigated the effect of AKI on the abundance and distribution of Prox-1+ cells at the mesoscale level using large scale three-dimensional (3D) imaging and tissue cytometry. ProxTom mice and their controls were subjected to ischemia-reperfusion (IR) or no surgery and kidneys were fixed on day 3. Large scale 3D imaging with confocal microscopy was done on 50mm thick sections spanning the entire cross section of the kidney, followed by tissue cytometry using the volumetric tissue exploration and analysis (VTEA) software tool.

Results

The average number of cells surveyed per specimen was 347,360 ± 36,647. Using VTEA, a gating strategy was devised to account for autofluorescence in the red channel, which was increased after IR due to cell debris and injury. IR samples displayed a significant increase in Prox-1+ cells compared to baseline controls: 717.2 ± 161.8 vs. 174.4 ± 62.1 Prox-1+ cells/mm3, respectively (p<0.05). In baseline controls, Prox-1+ cells were well-organized and predominately localized around large vessels in the hilum. However, after injury, the distribution of Prox-1+ cells shifted to the hilar parenchyma and inner medulla in a consistent pattern. Few cells could also be detected in the outer medulla and cortex.

Conclusion

We demonstrate a scale of analytics that is amenable to characterizing de novo renal LA during AKI, which informs the origin and distribution of renal LVs and the dynamics of LA. Such findings will enhance our understanding of the functional role of LVs during injury and help identify novel therapeutics for intervention in AKI.

Funding

  • NIDDK Support