Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

Abstract

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.

Novel Insights into Crystal-Induced Kidney Injury

“…They are reported to induce endothelial cell injury via complement activation [4]. Furthermore, they kill tubular epithelial cells by activating receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) [5, 6]. Activation of MLKL by RIPK3 results in disruption of the plasma membrane and a regulated form of necrosis, which is termed necroptosis [7].…”

“…Some of the lysosomal enzymes – e.g., cathepsin B – are reported to activate necroptosis by cleaving the necroptosis inhibitor protein RIPK1 in immune cells [26]. In fact, crystals of calcium oxalate, calcium phosphate, calcium pyrophosphate, cysteine, cholesterol, and MSU induced tubular cell necroptosis in vitro by engaging RIPK3 and pseudokinase MLKL [5, 6] (Fig. 2).…”

“…2). Moreover, both chemical inhibition as well as deficiency of promotors of necroptosis – for example, Ripk3 and Mlkl deficiency – protected mice from acute oxalate nephropathy [5]. On the contrary, deficiency of inhibitors of necroptosis – for example, repulsive guidance molecule b aggravated acute oxalate nephropathy [27].…”

“…Although crystals do not induce pyroptosis of tubular cells directly [5, 6], the chances of tubular cell death via pyroptosis cannot be ignored in crystalline nephropathies. Some inflammatory cytokines (e.g., TNFα) drive necroptosis of tubular cells to enhance oxalate crystal-induced AKI [5]. Multiple cathepsins are also suggested to regulate crystal-induced cell death in an inflammasome-independent manner [35].…”

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation

“…Lysosomal damage is necessary to trigger chromatin externalization by granulocytes (NETosis). NETosis is induced by various types of crystals (monosodium urate, calcium carbonate, and calcium oxalate) and requires peptidyl arginine deiminase-4 activity (10, 23) and two core proteins of the necroptosis pathway: RIPK3 and MLKL (24). To understand the biological interactions of NPs with granulocytes, engulfment, NETosis, and ROS production were further analyzed.…”