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Kidney Week

Abstract: PO0168

Endothelial Prolyl-Hydroxylase Domain Proteins Regulate Capillary Rarefaction Following Ischemic AKI and Reprogram Endothelial Metabolism

Session Information

  • AKI Mechanisms - 1
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Tiwari, Ratnakar, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
  • Kapitsinou, Pinelopi P., Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
Background

Endothelial cell (EC) metabolism has emerged as a new regulator of EC behaviour, but its role in capillary rarefaction, a common feature of progressive renal disease, remains unknown. Because EC sense oxygen and metabolic alterations through Prolyl hydroxylases 1 to 3 (PHD1-3), here we wished to define the impact of PHD inactivation on post-ischemic kidney injury outcomes, while in vitro studies focused on metabolic consequences.

Methods

Following the induction of renal ischemia-reperfusion injury (IRI), concurrent deletion of PHD1,2,3 was achieved by the Cdh5(PAC)CreER inducible system. Analysis was performed at day 14 post-IRI. Furthermore, we examined the impact of DMOG, a PHD inhibitor on angiogenic capabilities and global metabolic profiles of endothelial cells.

Results

Post-ischemic kidneys of PHD1,2,3ECKO showed more fibrosis, as indicated by 68% increase in collagen area (P=0.005) and significant upregulation of profibrotic genes Loxl2, Tgf-β and Acta2 (n=6-8, P<0.5) compared to controls. Quantitative analysis of endomucin staining showed 50% decrease in peritubular capillary density, associated with reduced endothelial proliferation as indicated by Ki-67 immunostaining (n=4, p=0.005). Notably, biochemical inactivation of PHDs by DMOG reduced EC proliferation in MTT assay (P= 0.0001) while cell cycle analysis showed decrease of cells in S (~39%, n=3, p=0.0007) and G2/M phase (~24%, n=3, p=0.04). Furthermore, DMOG reduced EC migration (50%, n=3, p=0.005) and tube formation. LC-MS analysis showed a profound effect of DMOG in glycolytic, TCA cycle, lipid and, nucleotide metabolites. Specifically, EC treated with DMOG showed an increase in lactate (1.46-fold, p<0.05) and significant reductions in citrate (2.2 fold, p<0.001), alpha-ketoglutarate (2.5 fold, p<0.001), fumarate (1.4-fold, p<0.05) and malate (1.3 fold, p<0.01). Supplementation with citrate partially rescued the proliferation defect induced by DMOG, suggesting that PHDs may affect angiogenic responses through alterations in mitochondrial metabolism.

Conclusion

Post-ischemic endothelial inactivation of PHDs promotes peritubular capillary rarefaction and fibrosis following AKI, a response which could involve alterations in mitochondrial metabolism.

Funding

  • NIDDK Support