Introduction: Sevelamer and polystyrene sulfonates are crystal-structured medications that are used to manage complications of end-stage kidney disease, such as hyperphosphataemia and hyperkalaemia in patients undergoing dialysis. When ingested, they bind to dietary phosphate or potassium, and thus prevent their gastrointestinal (GI) absorption. However, both sevelamer and polystyrene sulfonates commonly irritate the GI lining and can even cause colonic necrosis in severe cases. To date, little is known about the pathophysiology of crystal-induced GI lesions and the effect of these crystals on immune cells. We postulated that medicine crystals cause necroptosis in colonic epithelial cells depending on the crystal type and concentration, and that immune cells are more susceptible to crystal-induced cell death.
Methods: Colonic epithelial cell lines HCA7 and Caco2 as well as human blood neutrophils and monocytes were stimulated with different concentrations of sevelamer, polystyrene sulfonate and other crystals such as monosodium urate (MSU). Different cell death inhibitors including necrostatin 1, zvad and necrosulfonamide were tested. Cell death was quantified with flow cytometry (AnnexinV/PI) and LDH assays. The level of metabolic activity was determined by MTT assays and changes in mRNA expression of cytokines via RT-PCR. Furthermore, fluorescent microscopy was used to visualize human neutrophil and monocyte extracellular trap formation after crystal stimulation.
Results: Medicine crystals differ in shape and size compared to other previously investigated crystals such as MSU crystals. The metabolic activity of epithelial cells was reduced upon stimulation with different crystal types (HCA7; PBS vs 0.2mg/ml sevelamer: 100.0% vs 25.6%; p = 0.0001), whereas mRNA expression of TNF-α and NLRP3 were slightly upregulated. Interestingly, medicine crystals did not cause an increase in LDH release even at very high concentrations and the percentage of AnnexinV/PI positive cells remained also unchanged. On the other hand, sevelamer crystals caused an increase in LDH release in neutrophils, which was inhibited by necrostatin 1 (sevelamer 1mg/ml vs sevelamer 1mg/ml + 100µM nec1s: 38.3% vs 32.1% compared to positive control; p=0.005). Fluorescent microscopy showed that human neutrophils as well as monocytes underwent extracellular trap formation upon stimulation with medicine crystals.
Discussion: Our data show that medicine crystals temporarily reduce the metabolic activity and increase TNF-α and NLRP3 mRNA expression without inducing cell death in colon epithelial cells. In immune cells, however, sevelamer induced necroptosis as well as neutrophil and monocyte extracellular trap formation. Taken together, it seems that the gastrointestinal irritation is a secondary complication caused by the effect of medicine crystals on immune cells.