Abstract: SA-PO739
Protein-Bound Uremic Toxins Induce NLRP3 Inflammasome Activation in Proximal Tubule Cells
Session Information
- CKD: Mechanisms - III
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Mihajlovic, Milos, Utrecht Institute for Pharmaceutical Sciences, Utrecht,, UTRECHT, Netherlands
- Andreeva, Daria, Utrecht Institute for Pharmaceutical Sciences, Utrecht,, UTRECHT, Netherlands
- Steenhuis, Sonja, Utrecht Institute for Pharmaceutical Sciences, Utrecht,, UTRECHT, Netherlands
- Hilbrands, Luuk, Radboud University Medical Center, Nijmegen, Gelderland, Netherlands
- Masereeuw, Rosalinde, Utrecht Institute for Pharmaceutical Sciences, Utrecht,, UTRECHT, Netherlands
Background
Protein bound-uremic toxins (PBUTs) are not efficiently removed by hemodialysis in chronic kidney disease (CKD) patients. Their accumulation can lead to cellular dysfunction, inflammation and oxidative stress. Moreover, it has been shown that increased intrarenal expression of the NLRP3 receptor and IL-1β are associated with reduced kidney function, suggesting a critical role for the NLRP3 inflammasome in CKD progression. Here, we evaluated the effect of PBUTs on NLRP3 inflammasome activation in human conditionally immortalized proximal tubule epithelial cells (ciPTECs).
Methods
NLRP3 activation was studied after exposing cells to LPS, ATP, indoxyl sulfate (IS), and a mixture of eight anionic PBUTs (UT-mix). Expression levels of inflammasome components (NLRP3, caspase-1, IL-1β), IL-1β secretion, caspase-1 activity and production of reactive oxygen species (ROS) were evaluated.
Results
Exposure to a combination of LPS (1 µg/ml; 24h) and ATP (5 mM; 30 min) showed 3-fold (p<0.01) increase in IL-1β secretion, and 2-fold (p<0.05) increase in caspase-1 activity, suggesting that the NLRP3 pathway is functional in ciPTECs. Next, 24h exposure to increasing concentrations of IS increased mRNA expression of NLRP3 (2.3-fold; p<0.01), caspase-1 (2.2-fold; p<0.01) and IL-1β (24-fold; p<0.01). Similar results were observed for UT-mix: NLRP3 (1.7-fold increase; p<0.05), caspase-1 (1.8-fold increase; p<0.05) and IL-1β (4.5-fold increase; p<0.001). In addition, exposure to IS and UT-mix led to 10-fold (p<0.001) and 3-fold (p<0.001) increases in IL-1β secretion, respectively. Similarly, there was 1.9-fold (p<0.01) increase of caspase-1 activity upon treatment with IS and 1.8-fold increase after exposure to UT-mix. Furthermore, IS and UT-mix induced the production of intracellular ROS (2.6-fold increase for IS and 1.9-fold increase for UT-mix; p<0.05). Finally, IL-1β secretion was reduced when the N-acetylcysteine (1.8 mM; 24h) was added as a pre-treatment (47% reduction for IS and 35% reduction for UT-mix; p<0.05), suggesting that inflammasome activation is ROS-mediated.
Conclusion
PBUTs are able to induce NLRP3 inflammasome activation in proximal tubule cells via oxidative stress, suggesting their involvement in a local inflammatory response in kidney disease.
Funding
- Government Support - Non-U.S.