Abstract 16879: Endothelial Prolyl Hydroxylase 3 Regulates Post-Ischemic Kidney Injury Through HIF Dependent Mechanisms

Abstract

Introduction: Acute kidney injury (AKI) is highly prevalent clinical condition after cardiac surgeries and is associated with high morbidity and mortality in hospitalized patients. Ischemia reperfusion injury (IRI) is one of the major causes of AKI. Previously, we have reported a critical role for the endothelial oxygen sensor prolyl hydroxylase (PHD2) in ischemic AKI. Recently, we found that prolyl hydroxylase 3 (PHD3) shows robust increase in kidney endothelium after renal IRI. However, its role in ischemic AKI is unknown. Hypothesis: We hypothesized that endothelial PHD3 regulates post-ischemic kidney injury. Methods: To delete endothelial PHD3, VECadherin (Cdh5)-CreER transgenic mice were crossed to mice carrying conditional PHD3 allele, generating PHD3 iEC mice. To test the HIF-dependent role of PHD3, we generated Cdh5CreER; Phd3 flox/flox Arnt flox/flox mice ( PHD3ARNT iEC ). Inactivating ARNT (aryl hydrocarbon receptor nuclear translocator), also known as Hif-1β, blocks HIF signaling. Unilateral IRI was induced by renal artery clamping, and tamoxifen treatment was started at day 1 post IRI, followed by analysis at day 14. Results: Compared to Cre - controls, inactivation of Phd3 in endothelial cells (ECs) following IRI exacerbated kidney damage and inflammation, as indicated by increased expression of kidney injury molecule 1 ( Kim1 ) and profibrotic genes lysyl oxidase-like 2 ( Loxl2 ), transforming growth factor-beta 1 ( Tgfb1 ) , smooth muscle actin ( Acta2 ) and collagen deposition assessed by Picro Sirius red staining (n=6-7; p< 0.05). Post-ischemic inactivation of ARNT along with PHD3 ameliorated the effects of only PHD3 inactivation and normalized levels of Kim1 , Loxl2 , Tgfb1 and collagen deposition ( n=7; p< 0.05) . In vitro suppression of PHD3 in human primary EC using PHD3-siRNA showed significant upregulation of inflammatory molecules such as ICAM1 (Intercellular Adhesion Molecule 1), VCAM1 (Vascular Cell Adhesion Molecule 1), C-X-C motif chemokine ligand 9 and 10 ( CXCL9 and CXCL10 ), which were again blunted by ARNT- siRNA treatment (n= 3; p< 0.05). Conclusions: In summary, our data support a critical role for endothelial PHD3 in regulating post-ischemic kidney inflammation and fibrosis through HIF-dependent mechanisms.