Endothelial Prolyl Hydroxylase 3 (PHD3) Regulates Post-Ischemic Kidney Injury Repair and Inflammation

Background: Endothelial cells (EC) are prime targets for ischemia reperfusion injury (IRI) but the role of endothelial oxygen sensing in IRI remains poorly defined. Recently, we showed that post-ischemic concurrent inactivation of endothelial Phd1, Phd2 and Phd3 but not Phd2 alone promotes maladaptive kidney repair. Analysis of human and mouse scRNA-seq data suggested a potential role for the Phd1 and/or Phd3 isoforms in renal endothelial oxygen sensing. Here, we investigated the role of endothelial Phd1 and Phd3 in kidney repair following IRI. Methods: Post-ischemic inactivation of Phd1, Phd3 and Arnt (HIFβ) in ECs was achieved by the Cdh5(PAC)CreER. Analysis was performed on day 14 post-unilateral IRI. To inactivate PHD3 and ARNT genes in-vitro in ECs we used siRNA approach, while overexpression of PHD3 was achieved by lentivirus-based transduction. IFNγ was used to induce a pro-inflammatory response in-vitro. Results: Post-ischemic inactivation of endothelial Phd1 failed to alter post-IRI kidney repair, however, inactivation of endothelial Phd3 following IRI exacerbated kidney damage and fibrosis as indicated by significantly increased Kim1, Tgfβ, and Acta2 transcripts as well as enhanced collagen deposition and macrophage infiltration (n=5-6; p< 0.05), compared to Cre- controls. A cytokine array detected increase in multiple cytokines in the injured mutant kidney. Notably, post-ischemic concurrent deletion of endothelial Phd3 and Arnt restored kidney damage as shown by mRNA expression of pro-fibrotic genes and collagen deposition (n=6-7, p<0.05). In vitro, PHD3 knockdown enhanced IFNγ induced pro-inflammatory responses in EC based on mRNA expression of ICAM1, VCAM1, and other IFNγ responsive genes (n=3, p<0.05). Concurrent silencing of endothelial PHD3 and ARNT rescued the pro-inflammatory activation of ECs by IFNγ stimulation. Importantly, lentiviral PHD3 overexpression suppressed EC activation by IFNγ. Conclusion: Endothelial inactivation of PHD3 promotes post-ischemic AKI to CKD transition in a HIF dependent manner. Our studies a) identify PHD3 as a crucial regulator of post-ischemic kidney repair through mechanisms that involve regulation of IFNγ derived EC inflammation and b) have critical implications for the potential application of non-selective PHD inhibitors in ischemic AKI. Funding: NIDDK Support