The Role of P2X7 Receptor in Glomerulonephritis

[Speaker] Bahire Kalfaoglu:1
[Co-author] Tom Mckinnon:1, Charles D. Pusey:1, Frederick W. K. Tam:1, Theresa H. Page:1
1:Medicine, Imperial College London, UK

The ATP-gated P2X7 receptor (P2X7R) mediates activation of the NLRP3 inflammasome and the subsequent release of mature IL-1beta and IL-18. NLRP3 activation is linked to proteinuria and renal injury. Moreover, P2X7R deficiency is found to be renoprotective in the experimental models of glomerulonephritis (GN), suggesting a role for P2X7R as a therapeutic target for GN. Canonical P2X7R activity can be modulated by oligomerisation with 3 of its 10 splice variants (from A to J -A encoding the full-length receptor), but the expression and function of the remaining variants are yet to be described. This study aimed to determine the role of P2X7R and its splice variants in cytokine production and downstream events in cells involved in GN pathogenesis.

Cytokine levels were quantified by ELISA. Human renal cells, PBMCs and macrophages were stimulated with LPS followed by high millimolar ATP or BzATP +/- P2X7R antagonist A438079. Transcriptome profiles were analysed by RT-qPCR. Plasmid constructs or co-constructs containing P2X7R cDNAs were transiently expressed in HEK293Ts. The expression and functionality of the variants were examined by confocal microscopy, FACS and immunoblotting techniques.

Serum levels of IL-18 (p=<0.005), but not IL-18BP (p=<0.05), are elevated in GN in comparison to healthy subjects. To investigate the source of IL-18 we used LPS as a priming signal, followed by ATP stimulation. PBMCs and macrophages release IL-18 and IL-1beta in a P2X7R-dependent manner. Intriguingly, human primary podocytes upregulate NLRP3, PYCARD, IL-1beta and IL-18 mRNAs in response to LPS, but subsequent ATP stimulation does not induce IL-18 nor IL-1beta release. Mesangial cells and glomerular endothelial cells were also incapable of producing IL-18 under similar conditions. This was not due to the absence of P2X7R transcripts, as all splice variants mRNAs are detected in these cells. Furthermore, P2X7A/E co-construct transfected HEK293Ts show both intracellular and extracellular distribution.

Raised serum IL-18 in GN may originate from infiltrating mononuclear cells, but not podocytes, mesangial or glomerular endothelial cells. Studies of the P2X7R-E show for the first time that this variant can be expressed in HEK293Ts, and may be capable of modulating canonical receptor activity.

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