Abstract: TH-PO050
Microparticles Released in Response to Oxidative Stress from the Renal Epithelium Carry Active Neprilysin
Session Information
- AKI: Mechanisms - Primary Injury and Repair - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Campos, Begoña, University of Cincinnati, Cincinnati, Ohio, United States
- Bernardo, Sabrina Isabelle, University of Cincinnati, Cincinnati, Ohio, United States
- Heuker, Nicholas A., University of Cincinnati, Cincinnati, Ohio, United States
- Bockhorst, Samuel Paul, University of Louisville School of Medicine, Loveland, Ohio, United States
- Thakar, Charuhas V., University of Cincinnati, Cincinnati, Ohio, United States
Background
Acute kidney injury (AKI) is associated with significant morbidity, including remote organ dysfunction. In response to stress, cells release phenotypically and quantitatively distinct microparticles (MP). MP are microvesicles (1000 nm) derived from several cell types and released in response to stress or injury. Neprilysin (CD10), is a membrane-bound zinc activated endopeptidase, richly expressed in the renal proximal tubular epithelial cell (RPTEC) brush border. Neprilysin catalyzes the degradation of endogenous vasodilator/natriuretic peptides suggesting that its physiological action modulates the hemodynamic balance. Whether MP containing neprilysin released by renal epithelium can be mediators of biological activity is not known.
Methods
Human RPTEC immortalized cell line was used. Cells were exposed to 0.03 molar H2O2 for 1 hour and compared to controls in 3 or more different sets of experiments. We evaluated the levels of neprilysin using an ELISA assay and enzymatic activity with a fluorometric assay. Human samples (citrated plasma) were tested for the presence of biologically activity (Neprilysin levels and peptidase assay) derived from prospectively collected samples in AKI cases and controls.
Results
We have shown that neprilysin is present in RPTEC using immunofluorescence. We also reported that under conditions of oxidative stress the level of released MP is significantly different when compared to controls. To evaluate if the released MP are biologically active, we assessed the protease activity characteristic of neprilysin. The results showed that the maximal activity of neprilysin was in healthy cells and was 5-fold lower after treatment with oxidative stress. We then evaluated the protease activity in MP released from RPTEC under the same conditions. Our results showed that the peptidase activity was present and the activity correlated directly with the protein concentration. In a pilot of 30 cases and controls of AKI, we were able to measure the levels of plasma neprilysin using the ELISA assay as well as the confirm the peptidase activity in the MP derived from human samples.
Conclusion
The release of Neprilysin in microparticles derived from renal tubular epithelial cells are functionally active, and could serve as a biological link between epithelium and micro-vasculature
Funding
- Clinical Revenue Support