P0087TRAF3 MODULATION: A NOVEL MECHANISM OF THE ANTI-INFLAMMATORY EFFECTS OF VITAMIN D RECEPTOR AGONIST PARICALCITOL IN RENAL DISEASE

Abstract

Abstract Background and Aims Tumor necrosis factor receptor-associated factors (TRAFs) are critical signaling adaptors downstream of pro-inflammatory receptors, involved in canonical and non-canonical nuclear factor-κB (NF-κB) activation. Vitamin D receptor agonists (VDRAs) exert beneficial effects in renal disease and possess anti-inflammatory properties, but the underlying mechanism remains unknown. Our aim was to investigate TRAF3 involvement on renal disease and its potential modulation by VDRAs. Method Studies were done in isolated peripheral blood mononuclear cells (PBMCs) from end-stage renal disease (ESRD) on haemodialysis (treated or not with VDRAs) patients, healthy donors or cultured renal cells. Preclinical models of renal damage were: unilateral ureteral obstruction (UUO), acute renal damage induced by Folic acid (FA) administration and TWEAK-induced renal inflammation. The effect of VDRA (paricalcitol) was tested in vitro and in vivo. Results In PBMCs isolated from ESRD, TRAF3 protein levels were downregulated compared to healthy controls, associated to activation of non-canonical NF-κB2 (downregulation of RelB or p52/NF-κB2 protein levels) and altered pro-inflammatory cytokine profile. Interestingly, treatment of those patients with the VDRA paricalcitol restored all the described features. In vitro experiments in PBMCs and tubular epithelial cells exposed to inflammatory stimuli showed that paricalcitol prevented TRAF3 downregulation, NF-κB2 activation and proinflammatory genes upregulation. Moreover, immunoprecipitation studies showed that paricalcitol diminished TRAF3 ubiquitination in the K48-linked chains and CIAP1-TRAF3 interaction. TRAF3 overexpression by CRIPS/cas9 technology mimics VDRAs effects. In preclinical models of renal damage paricalcitol inhibited renal NF-κB2 activation, reducing p52 and RelB nuclear accumulation and transcriptional activity associated to lower renal inflammation. In TWEAK-induced renal injury in VDR knockout mice, paricalcitol prevented TRAF3 downregulation and NF-κB2-dependent gene upregulation, suggesting a VDR-independent anti-inflammatory effect of paricalcitol. Conclusion The present studies identify TRAF3 downregulation as a key feature promoting inflammation in CKD identifying non-canonical NF-κB activation as a key driver of inflammation in this context. The fact that the VDR is not required for paricalcitol actions suggests novel-signaling pathways involved in the anti-inflammatory effect of paricalcitol. This may allow the design of drugs that restore TRAF3 levels but are devoid of VDR-dependent effects on mineral bone metabolism, which may limit the use of VDRAs as anti-inflammatory agents.