Kidney Week

Abstract: FR-OR028

Inactivation of Endothelial HIF Polyhydroxylases Following Ischemic AKI Promotes Kidney Fibrosis

Session Information

• AKI: Mechanisms - Inflammation and AKI-CKD Transition
  November 08, 2019 | Location: 202, Walter E. Washington Convention Center
  Abstract Time: 05:54 PM - 06:06 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Tiwari, Ratnakar, KUMC - University of Kansas Medical Center, Kansas City, Kansas, United States

Ratnakar Tiwari, PhD



Dr Tiwari was awarded PhD in Biochemistry in 2018 from Babu Banarasi Das University, Lucknow, India. He is currently working as a post-doctoral research fellow at the Jared Grantham Kidney Institute of University of Kansas Medical Center, Kansas USA. Dr Tiwari has a broad background in toxicology and molecular biology with specific training in chronic kidney disease and metabolic disorders. He has been working on kidneys diseases such as minimal change disease, diabetic nephropathy, acute kidney injury and chronic kidney diseases. Dr Tiwari has coauthored 9 research articles and has been awarded several awards such as NIEHS travel award (2014), best poster award (STOX-India 2015) and K-INBRE postdoctoral grant 2019.

• Schonfeld, Michael P., KUMC - University of Kansas Medical Center, Kansas City, Kansas, United States

Michael P. Schonfeld,

• Kapitsinou, Pinelopi P., KUMC - University of Kansas Medical Center, Kansas City, Kansas, United States

Pinelopi P. Kapitsinou,

Background

Key regulators of hypoxic vascular responses are Hypoxia-Inducible-Factors (HIF)-1 and -2, transcription factors whose activity is negatively regulated by prolyl-hydroxylase domain proteins 1 to 3 (PHD1 to PHD3). Little is known about endothelial cell (EC) specific functions of PHDs in response to acute kidney injury (AKI), a common problem associated with significant morbidity and mortality. Here, we used a genetic approach to investigate the function of endothelial PHDs in renal ischemia-reperfusion injury (IRI).

Methods

Cdh5(PAC)CreER^T2 inducible system was used to induce conditional deletion of PHD1,2,3 in ECs (Cdh5(PAC)CreER^T2; Phd1^f/fPhd2^f/fPhd3^f/f referred as EC-PHD1/2/3 mutants) while the recombination efficiency was assessed by crossing Cdh5(PAC)-CreER ^T2 transgenic mice to ROSA26-ACTB-tdTomato,-EGFP reporter mice. Mice were subjected to unilateral IRI and at Day 1 post IRI they were started on tamoxifen (total of 4 doses) to inactivate PHDs. Samples were collected at day 14 after IRI.

Results

The Cdh5(PAC)CreER^T2 showed efficient recombination in the kidney endothelium based on FACs analysis and imaging of kidney tissue from the reporter mice. Immunofluorescence analysis of PHDs showed persistent expression of PHDs following IRI. Post-ischemic inactivation of endothelial PHDs (1-3) exacerbated infiltration of inflammatory cells and tubular damage at day 14 after IRI assessed by histopathological analysis of injured kidneys. EC-PHD1/2/3 mutants showed increased expression levels of profibrotic genes Loxl2 (n=6-8, P= 0.03). and Tgf-β (n=6-8, P= 0.005). Significant deposition of matrix in interstitial spaces was observed in kidneys of EC-PHD1/2/3 mutants compared to Cre-controls as indicated by Sirius red staining. Transmission electron microscopic examination showed prominent endothelial cell damage in the kidney of EC-PHD1/2/3 mutants, which was associated with peritubular capillary rarefaction as indicated by endomucin staining. Furthermore, treatment of human ECs with a PHD inhibitor following the induction of hypoxia-reoxygenation led to significant suppression of EC-proliferation.

Conclusion

Our data show a critical role for endothelial PHDs following ischemic AKI. Inactivation of endothelial PHDs following ischemic AKI promotes kidney inflammation, peritubular capillary rarefaction and fibrosis.

Funding

• NIDDK Support