Abstract: TH-PO207
ABCA1 Deficiency Primes Inflammasome via APE1/IRF1 Axis in Podocytes
Session Information
- Diabetic Kidney Disease: Basic - I
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Authors
- Ito, Marie, University of Miami School of Medicine, Miami, Florida, United States
- Varona Santos, Javier T., University of Miami School of Medicine, Miami, Florida, United States
- Gurumani, Margaret Zvido, University of Miami School of Medicine, Miami, Florida, United States
- Sloan, Alexis J., University of Miami School of Medicine, Miami, Florida, United States
- Merscher, Sandra M., University of Miami School of Medicine, Miami, Florida, United States
- Fornoni, Alessia, University of Miami School of Medicine, Miami, Florida, United States
Background
Decreased ATP Binding Cassette Transporter A1 (ABCA1) expression and Caspase-4-mediated noncanonical inflammasome have been described in diabetic kidney disease (DKD). However, a direct link between these two pathways as not been established.
Methods
To investigate the role of ABCA1 deficiency in inflammasome, we stably transfected human podocytes with scrambled siRNA as a negative control (siCO) and siABCA1 RNA (siABCA1). Podocytes with transient siRNA knockdown of Interferon regulatory factor 1 (IRF1) were used to study the role of IRF1 in inflammasome. Podocytes were treated with TAK-242 (TLR4 inhibitor) or APX3330 (APE1 redox inhibitor) to investigate the role of toll-like receptor 4 (TLR4) and Apurinic/apyrimidinic endonuclease 1 (APE1) in IRF1 regulation. The nuclear fractions of siCO and siABCA1 podocytes were isolated using the ab109719 cell fractionation kit and protein levels of APE1 were evaluated by Western blot analysis. mRNA and protein levels of IRF1 and inflammasome-related genes (NLR family pyrin domain containing 3 (NLRP3), Caspase-4, Gasdermin D, Caspase-1 and Interleukin 1β (IL1β)) were quantified by Western blot analysis and real-time PCR.
Results
In siABCA1 podocytes, mRNA levels of IRF1, Caspase-4, GSDMD, Caspase-1 and IL1β but not of NLRP3 and protein levels of Caspase-4, GSDMD and IL1β were significantly increased compared to siCO podocytes. Since IRF1 regulates the expression of Caspase-4, GSDMD and/or Caspase-1 in macrophages, we conducted siRNA knockdown of IRF1 and find that IRF1 knockdown in siABCA1 podocytes prevented increases in Caspase-4, GSDMD and IL1β expression. As candidate mechanism in siABCA1-mediated IRF1 regulation, we next investigated the role of TLR4, which was shown by others to mediate NLRP3 activation in ABCA1 deficient proximal tubular cells, and of APE1, which is excreted from cells via ABCA1. Whereas TAK-242, did not decrease mRNA levels of IRF1 and Caspase-4, APX3330 abrogated siABCA1-inducued expression of IRF1 and Caspase-4. APE1 protein expression was also increased in the nuclear fraction of siABCA1 podocytes compared to siCO.
Conclusion
These data indicate that ABCA1 deficiency in podocytes caused nuclear APE1 accumulation, which reduces transcription factors to increase the expression of IRF1 and IRF1 target inflammasome-related genes, leading to proptosis priming.
Funding
- Other NIH Support