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Published on: September 1, 2015
Functional expression of the damage-associated molecular pattern receptor P2X7 on canine kidney epithelial cells
Iman Jalilian1, Mari Spildrejorde, Aine Seavers
1School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
Abstract:
Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin-Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5'-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2'(3')-O-(4-benzoylbenzoyl)ATP induced ethidium(+) uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.
Insights
Madin-Darby canine kidney cells express functional P2X7 receptors, crucial for inflammation and immunity. This finding aids research into kidney disorders in dogs and humans.
Area of Science:
- Immunology
- Cell Biology
- Renal Science
Background:
- Epithelial cells play a key role in immune responses and inflammation.
- P2X7 receptors bind extracellular adenosine 5'-triphosphate (ATP), a damage-associated molecular pattern.
- Understanding epithelial cell receptors is vital for studying renal disorders.
Purpose of the Study:
- To investigate the expression and functionality of P2X7 receptors in Madin-Darby canine kidney (MDCK) cells.
- To determine if MDCK cells express other key immune-related molecules like Toll-like receptor 4 and NALP3 inflammasome components.
- To establish MDCK cells as a model for studying epithelial cell involvement in renal inflammation.
Main Methods:
- Reverse transcription (RT)-PCR and immunoblotting were used to detect P2X7 receptor expression.
- Flow cytometry assessed ethidium(+) uptake, a measure of P2X7 receptor activation, in response to ATP.
- P2X7 receptor antagonists (KN-62, A438079) were used to confirm P2X7-mediated responses.
Main Results:
- RT-PCR and immunoblotting confirmed the expression of P2X7 receptors in MDCK cells.
- ATP stimulation induced ethidium(+) uptake in MDCK cells, indicating functional P2X7 receptors.
- This ATP-induced uptake was significantly inhibited by P2X7-specific antagonists.
- MDCK cells also expressed Toll-like receptor 4, NALP3, caspase-1, IL-1β, and IL-18.
Conclusions:
- The MDCK epithelial cell line expresses functional P2X7 receptors.
- MDCK cells possess components of the innate immune system, including TLR4 and the NALP3 inflammasome.
- This cell line serves as a valuable tool for investigating the role of these molecules in kidney epithelial cells and associated renal disorders in both canine and human contexts.
