Modeling Kidney Proximal Tubular Crystallopathy In Vitro

Background: Kidney crystallopathy can be characterized by the presence of crystals within renal tubules, leading to obstruction, cytotoxic effects, and inflammatory responses. Here, a novel proximal tubule cell model was developed to investigate the mechanisms underlying toxicant-induced crystal formation and renal inflammation. Methods: Conditionally immortalized proximal tubule epithelial cells (ciPTECs) were used to investigate the role of uric acid, a known crystal-forming metabolite, and the influence of pH on crystal formation. A series of in vitro assays were used to assess cytotoxicity, oxidative stress, mitochondrial function, inflammation as well as the expression of NLRP3 inflammasome related markers on the protein level. Results: Exposing ciPTECs to uric acid decreased cell viability, which was more pronounced under reduced pH conditions with a significant reduction starting at 200 μg/ml at pH 5.5 vs 800 μg/ml at pH 7.4. Various shapes of uric acid crystals were formed (Figure 1). Reactive oxygen species production was increased at 200 μg/ml at all pH levels tested (viz. 5.5, 6.0 and 7.4). Furthermore, uric acid induced the expression of the inflammasome-related markers caspase-1, ASC, TNFα at the mRNA level, and IL-1β at the protein level. In line with cell viability, these effects were most pronounced with higher uric acid concentrations and at lower pH levels. Conclusion: A novel model was established simulating kidney crystallopathy and its impact on inflammasome components, caspase activity, cytokine production, and the NF-κβ pathway. This model will be used to study crystallopathy in more detail to provide an adverse outcome pathway for crystal-forming nephrotoxicants. This project received funding from the European Union’s Horizon 2020 research and innovation programme (GA No 963845, ONTOX). Funding: Government Support – Non-U.S.Figure 1. Uric acid crystals formed after 72h incubation with A) 800 μg/ml at pH 5.5 and B) 600 μg/ml at pH 5. 10x magnification.