TLR4 and NALP3 inflammasome in the development of endothelial dysfunction in uraemia Susana Martin-Rodriguez *,a , Carolina Caballo *,a , Gabriela Gutierrez * , Manel Vera † , Josep M. Cruzado ‡ , Aleix Cases † , Gines Escolar * and Maribel Diaz-Ricart * * Department of Hemotherapy-Hemostasis, Institut d’Investigacions Biom ediques August Pi i Sunyer (IDIBAPS), Hospital Clinic, Universitat de Barcelona, Barcelona, Spain, † Department of Nephrology, Institut d’Investigacions Biom ediques August Pi i Sunyer (IDIBAPS), Hospital Clinic, Universitat de Barcelona, Barcelona, Spain, ‡ Diaverum Institut Hemodi alisis, Barcelona, Spain ABSTRACT Background The increased cardiovascular risk present in chronic kidney disease (CKD) is related to the development of endothelial dysfunction, whose mechanisms are still unclear. Accumulation of toxins and pro- inflammatory cytokines may constitute danger-associated molecular patterns (DAMP) to which endothelial cells are continuously exposed. Potential involvement of mechanisms recognizing DAMP, such as TLR and inflam- masomes, has been explored. Materials and methods Endothelial cells in culture were exposed to sera samples collected from patients with CKD: (i) stages 4–5 not on dialysis (PreD), (ii) on maintenance haemodialysis (HD) and (iii) peritoneal dialysis (PD). Changes in TLR4 and ICAM-1 expression, reactive oxygen species (ROS) production and TLR4 signalling were explored. Assembly of NALP3 inflammasome components was also investigated. Results TLR4 was expressed at the cell surface and increased significantly in response to PreD, HD and PD sera, paralleling with the activation of the cell stress protein Akt and the inflammation-related transcription factor NFjB, with elevated surface ICAM-1 expression and ROS production. TLR4 blockade partially decreased these effects. Exposure of cells to uraemic sera induced assembly of NALP3 components, with caspase-1 activation, especially in response to HD and PD sera. Conclusions TLR4 and NALP3 inflammasomes, crucial elements of innate immunity, contribute to the devel- opment and perpetuation of endothelial dysfunction in response to the uraemic toxicity. These mechanisms constitute potential therapeutic targets to improve endothelial dysfunction and to reduce the increased car- diovascular risk in CKD. Keywords Chronic kidney disease, endothelial dysfunction, innate immunity, NALP3, TLR4, uraemia. Eur J Clin Invest 2015; 45 (2):160–169 Introduction The pathophysiology of the enhanced atherothrombotic risk in CKD is complex. In vivo and in vitro evidence indicates the coexistence of endothelial dysfunction together with a chronic inflammatory state and oxidative stress in uraemic patients. In the development of these pathological processes, there are at least two components: a humoral and a cellular one. The humoral component consists of the presence of uraemic toxins [1,2] and factors released by activated blood cells [3,4]. Uraemic toxins are accumulated in the sera of patients with CKD and alter the function of the different blood cell populations, involved in haemostasis and atherosclerosis that release cytokines enriching the humoral component. In response to cytokines, endothelial cells produce chemokines, pro-inflam- matory cytokines and also express adhesion molecules, pro- moting the adhesion of circulating leucocytes that translocate into the intima, initiating the atherosclerotic process. Although endothelial dysfunction in CKD has been well characterized before, the precise molecular mechanisms potentially involved remain to be fully elucidated. Factors inducing endothelial dysfunction in CKD include pulsatile blood flow and shear stress, oxidized LDL particles, hyperphosphatemia, reactive oxygen species, advanced glycation end products, microbial-derived products, endogenous damage-associated molecules and circulating a These authors contributed equally to the manuscript 160 ª 2014 Stichting European Society for Clinical Investigation Journal Foundation DOI: 10.1111/eci.12392 ORIGINAL ARTICLE