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Kidney Week

Abstract: SA-PO179

β2-Integrins Are Indispensable for HIF1α Activation in Neutrophils by Controlling Translational Initiation Factor Phosphorylation

Session Information

  • AKI: Mechanisms - III
    November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Kling, Lovis, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany
  • Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Jerke, Uwe, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Rousselle, Anthony, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Ivanov, Andranik, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Core Unit Bioinformatics, Berlin, Germany
  • Eckardt, Kai-Uwe, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany
  • Schreiber, Adrian, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany
  • Kettritz, Ralph, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany
Background

In several inflammatory kidney diseases, circulating blood neutrophils migrate to sites with high cytokine but low oxygen concentrations. Migrating neutrophils interact with extracellular matrix proteins via β2-integrins. We hypothesized that β2-integrins control hypoxia-inducible factor 1α (HIF1α) activation under basal and inflammatory conditions.

Methods

HIF1α in human neutrophils was studied by immunoblotting, qPCR, bulk mRNA sequencing. Neutrophil suspension was achieved on polyhema, and adhesion on fibronectin (FN). Prolyl hydroxylases (PH) were inhibited with Roxadustat (ROX) and normobaric hypoxia (1% O2). β2-integrin signaling was analyzed by blocking and activating antibodies.

Results

We observed HIF1α protein in ROX-, but not GMCSF-treated neutrophils after 4h with a 3-fold synergistic effect by combined GMCSF/ROX treatment. Increased HIF1α protein was observed in neutrophils cultured on FN but not under stringent suspension conditions on polyhema. Importantly, HIF1α protein in combined ROX/GMCSF-treated neutrophils on FN was strongly reduced by blocking β2-integrins and, conversely, induced by activating β2-integrin antibodies on polyhema. The synergistic ROX/GMCSF effect as well as the blocking β2-integrin effect for HIF1α protein were recapitulated under hypoxic conditions. Mechanistically, the HIF1α mRNA increase by GMCSF was necessary but not sufficient to explain the adhesion-dependent HIF1α protein induction. Importantly, GMCSF increased serine phosphorylation of eIF4E and 4EBP1 HIF1α translation initiation factors in neutrophils on FN in an β2-integrin dependent manner. Synergistic HIF1α protein induction was demonstrated in neutrophils that transmigrated through FN towards GMCSF and ROX in vitro. Neutrophil bulk mRNA sequencing revealed HIF1α-dependent enrichment of cytoskeletal gene sets under inflammatory conditions. Finally, PH inhibition by ROX or hypoxia enhanced cytoskeleton-dependent neutrophil adhesion.

Conclusion

β2-integrin engagement restricts HIF1α activation to neutrophils that emigrate from the blood to inflammatory sites under pharmacological or hypoxic conditions.

Funding

  • Government Support – Non-U.S.