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Abstract: FR-PO154

Uromodulin and Injury Severity Alter Neutrophil Diversity and Signaling in AKI

Session Information

  • AKI: Mechanisms - II
    November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Winfree, Seth, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • LaFavers, Kaice Arminda, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Khan, Shehnaz, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Micanovic, Radmila, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Melo ferreira, Ricardo, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Cheng, Ying-Hua, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Eadon, Michael T., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

Neutrophils are central to the pathogenesis of acute kidney injury (AKI). Neutrophil subtypes have been described in infection, cancer, and sterile inflammation. Little is known about the role of neutrophil subtypes in AKI. The kidney is uniquely poised to affect neutrophil subtypes via Uromodulin (UMOD)-a protein made exclusively in the thick ascending limb and modulator of granulopoiesis. To explore a role for neutrophil subtypes and the impact of UMOD in AKI, we interrogated kidneys after ischemic injury in wild-type (WT) and UMOD knock-out (KO) mice with mesoscale confocal microscopy and single-cell and spatial transcriptomics.

Methods

Kidneys from WT or UMOD KO mice after Sham or 22- or 30-min ischemic injury were frozen in OCT for spatial transcriptomics, fixed for microscopy, or processed for single-cell RNASeq (whole kidney and CD45+ enriched cell suspensions). Sequencing results were processed with CellRanger and analyzed in Seurat (cell types), ReactomePA (pathways), Monocole (trajectories) and SPOTLight (deconvolution). Confocal microscopy was performed on whole kidney sections using the nuclear stain DAPI and markers of neutrophils, transcription factors and injury and cells were quantitated with tissue cytometry (Volumetric Tissue Exploration and Analysis).

Results

Neutrophil localization in the kidney in AKI was sensitive to injury severity and absence of UMOD. Transcriptionally, 7 distinct neutrophil subtypes were uncovered. Further, the proportions and pathway activation of these subtypes and neutrophil transcriptional trajectories were sensitive to the severity of ischemic injury. In severe injury there was an increase in putative immature neutrophils and CXCL3 expression in neutrophils. In injury of UMOD KO mice, neutrophils were retained in the cortex and CXCR2 ligands were uniquely upregulated in proximal tubule epithelia.

Conclusion

Our study uncovers an underappreciated diversity of neutrophil subtypes in AKI that is sensitive to injury severity and UMOD. We demonstrate unique subtypes of neutrophils in AKI correlated with changes in neutrophil localization and concomitant alterations in the CXCR2 signaling axis. Thus, our results suggest the neutrophil response in AKI is nuanced and modulated by injury severity and UMOD via the CXCR2 signaling axis.

Funding

  • Veterans Affairs Support