Introduction: Cadmium (Cd) is a heavy metal, which affects the important organs, such as liver, kidney, spleen, lung, and heart. Cadmium mostly accumulated within the kidneys and impairs it. The mechanisms underlying cadmium nephrotoxicity are not fully understood. Ceramides play important roles in many cellular processes, such as differentiation, cell proliferation, and apoptosis. This study was designed to evaluate the possible protective effects of ARC39 and GW4869 against Cd-induced renal toxicity by reducing the ceramide.

Methods: LLC-PK-1 cells were cultured DMEM supplemented with 10% FBS, 1% (v/v) penicillin and streptomycin. To evaluate ceramide level after exposure of cadmium and SMase inhibitors, LLC-PK1 cells were seeded with 1x10⁶ cells/well into 6-well plates, and treated with Cd (20 μM), Cd+ARC39 (20 and 10 μM, respectively), Cd+GW4869 (20 and 10 μM, respectively) and incubated for 24 h. After collection the samples, 100 uL of cell lysate were extracted by chloroform:methanol (2:1) mixture. The chloroform phase was dried at a vacuum evaporator and the dry residue was reconstituted in 100 µl MeOH before injecting into LC-MS/MS system. Ceramides were eluted by C18 column (150 mm × 2 mm × 3 μm; Phenomenex), with gradient elution condition generated from mobile phase A (10 mM ammonium acetate in water with 0.1% formic acid) and mobile phase B (10 mM ammonium acetate in ACN:Propanol-2 (4:3; v/v) with 0.1% formic acid). The mass spectrometer was operated in multiple reaction monitoring positive ionization mode, monitored the following transitions: m/z 538.2>264.3 for Cer16, 552.2>264.3 for Cer17, 548.2>264.3 for Cer18, 622.2>264.3 for Cer22, 650.4>264.3 for Cer24 and 648.4>264.3 for Cer24:1. The concentration of each ceramide was calculated according to calibration curves using the peak-area ratio.

Results: Exposure to 20 μM Cd for 24 h induced formation of cellular ceramides (C16:0, C18:0, C22:0, C24:0, C24:1) the cadmium group compared to untreated group. Data demonstrate that the long-chain ceramide C24:1 revealed the highest proportion of total ceramide. Each of ceramide and total ceramide effectively inhibited by pre-incubation with ARC39 compound at a concentration 10 μM. Total ceramide levels of GW4869 group (10 μM) were obviously dropped in comparison to the cadmium group and control groups. On the other hand, the S1P level was not considerably affected. Moreover, exposure of the cells to ARC39 and GW4869 increased cell viability within 24 hours of culture.

Conclusion: In conclusion, our results reveal that incubation of LLC-PK-1 cells with cadmium for 24 h increased the ceramide level and reduced the cell viability and induced the DNA damage in in vitro experiments. The alteration of ceramide may be considered as a biomarker of cadmium induced kidney failure and pretreatment with ARC39 and GW4869 may prevent the Cd-induced damage by inhibition of SMase.

Acknowledgment: This research was supported by "Regional Innovation Strategy (RIS)" through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(MOE)(2021RIS-001).