Supplementary Material for: Induction of vesicular trafficking and JNK-mediated apoptotic signaling in mononuclear leukocytes marks the immuno-proteostasis response to uremic proteins.

Introduction: Uremic retention solutes have been alleged to induce cell death in different cell types, including peripheral blood mononuclear leukocytes (PBL), which may contribute to uremic leukopenia and immune dysfunction. Methods: The molecular effects of these solutes were investigated in uremic PBL (u-PBL) and mononuclear cell lines (THP-1 and K562) exposed to the high-molecular-weight fraction of uremic plasma (u-HMW; prepared 50 kDa microconcentrator ultrafiltration). Results: u-PBL show reduced cell viability and increased apoptotic death compared to healthy control PBL (c-PBL). u-HMW induce apoptosis both in u-PBL and c-PBL, as well as in mononuclear cell lines, also stimulating cellular H2O2 formation and secretion, IRE1-α-mediated endoplasmic reticulum stress signaling and JNK/cJun pathway activation. Also, u-HMW induce autophagy in THP-1 monocytes. u-PBL were characterized by the presence in their cellular proteome of the main proteins and carbonylation targets of u-HMW, namely albumin, transferrin and fibrinogen, and by the increased expression of RAGE, a scavenger receptor with promiscuous ligand binding properties involved in leukocyte activation and endocytosis. Conclusions: In conclusion, large uremic solutes induce abnormal endocytosis and terminal alteration of cellular proteostasis mechanisms in PBL, including UPR/ERSR and autophagy. These are potent triggers of JNK-mediated apoptotic signaling. The findings in this study describe at the molecular level the suicidal role of immune cells in facing the uremic disturbances of systemic proteostasis, a process that we define as the immuno-proteostasis response of uremia.