Hydrogen sulfide attenuates uranium-induced kidney cells pyroptosis via upregulation of PI3K/AKT/mTOR signaling.

We have identified that hydrogen sulfide (H2 S), a gaseous mediator, plays a crucial role in antioxidative, anti-inflammatory, and cytoprotective effects on uranium (U)-triggered rat nephrotoxicity. Pyroptosis is a special mode of inflammation and programmed cell death involved in the activation of inflammasome and Caspase-1 and the release of inflammatory cytokines. This study aims to confirm whether H2 S can alleviate U-induced rat NRK-52E cell pyroptosis and to investigate the H2 S underlying regulatory mechanism. Our results indicate that pretreatment with NaHS (an H2 S donor) significantly inhibited U-increased reactive oxygen species level, NLRP3, apoptosis-related speck-like protein consisting of a caspase recruitment domain (ASC), and cleaved Caspase-1 proteins expression, gasdermin D messenger RNA (GSDMD mRNA) expression, interleukin (IL)-1ß and IL-18 contents, lactate dehydrogenase leakage, and numbers of double-positive dying kidney cells. NaHS application evidently augmented phosphorylated PI3K, AKT, and mTOR expression as well as ratios of their respective phosphorylation to the corresponding total proteins which were downregulated by U treatment. But, LY294002 (a PI3K inhibitor) administration effectively abrogated the consequences of NaHS on the levels of p-PI3K, cleaved Caspase-1, ASC and NLRP3 proteins, GSDMD mRNA expression, and (IL)-1ß and IL-18 contents. Simultaneously, LY294002 significantly reversed the effects of NaHS on U-induced pyroptosis rate and cytotoxicity. Taken together, these results indicate that H2 S ameliorated U-triggered NRK-52E cells pyroptosis via upregulation of PI3K/AKT/mTOR pathway, suggesting a novel role for H2 S in the management of nephrotoxicity caused by U exposure.

Uranium induces kidney cells apoptosis via reactive oxygen species generation, endoplasmic reticulum stress and inhibition of PI3K/AKT/mTOR signaling in culture.

Uranium (U) induces generation of excessive intracellular reactive oxygen species (ROS), which is generally considered as a possible mediator of U-triggered kidney tubular cells injury and nephrotoxicity. Our goal is designed to elucidate that the precise molecular mechanism in ROS downstream is association with U-induced NRK-52E cells apoptosis. The results show that U intoxication in NRK-52E cells reduced cell activity and triggered apoptosis, as demonstrated by flow cytometry and apoptotic marker cleaved Caspase-3 expression. U exposure triggered endoplasmic reticulum (ER) stress, which is involvement of apoptosis determined by marker molecules including GRP78, PERK, IRE1, ATF6, CHOP, cleaved Caspase-12, and Caspase-3. Administration of antioxidant N-acetylcysteine (NAC) effectively blocked U-triggered ROS generation, ER stress, and apoptosis. U contamination evidently decreased the expression of phosphorylation PI3K, AKT, and mTOR and ratios of their respective phosphorylation to the corresponding total proteins. Application of a PI3K activator IGF-1 significantly abolished these adverse effects of U intoxication on PI3K/AKT/mTOR signaling and subsequently abrogated U-triggered apoptosis. NAC also effectively reversed down-regulation of phosphorylated PI3K induced by U exposure. Taken together, these data strongly suggest that U treatment induces NRK-52E cells apoptosis through ROS production, ER stress, and down-regulation of PI3K/AKT/mTOR signaling. Targeting ROS formation-, ER stress-, and PI3K/AKT/mTOR pathway-mediated apoptosis could be a novel approach for attenuating U-triggered nephrotoxicity.

Uranium induces kidney cells pyroptosis in culture involved in ROS/NLRP3/caspase-1 signaling.

Pyroptosis is an exceptional mode of inflammation and programmed cell death involved in inflammasomes and Caspase-1 activation and inflammatory cytokines releasing. Our goal is to explore whether uranium (U)-intoxication could induce NRK-52E cells pyroptosis in vitro and its underlying molecular mechanism. Rat NRK-52E cells were intoxicated with U concentrations (400-500 µM) for 24 h. The results indicate that the cells showed characteristic features of pyroptosis, which were identified through augmented NLRP3 and cleaved Caspase-1 proteins expression, GSDMD mRNA level, mature interleukin IL-18 and IL-1ß contents, LDH leakage, and the number of double-positive cells. But, administration of glycine (an inhibitor of pyroptosis) effectively attenuated U-induced pyroptosis, LDH releasing and cytotoxicity. Pretreatment of CRID3 (an inhibitor of NLRP3 inflammasome) evidently abrogated NLRP3 and cleaved Caspase-1 proteins and GSDMD mRNA expression which all were up-regulated by U exposure. Simultaneously, CRID3 significantly reversed U-increased pyroptosis rate and active interleukin IL-18 and IL-1ß contents. NAC application (an ROS scavenger) effectively decreased U-increased ROS content and NLRP3 expression and restored U-induced pyroptosis. Taken together, our results suggest that U-treatment can trigger NRK-52E cells pyroptosis which is involvement of ROS/NLRP3/Caspase-1 pathway. Targeting ROS/NLRP3/Caspase-1-mediated pyroptosis may be a novel approach for attenuating U nephrotoxicity.