The mechanism of oxalate-induced nucleotide binding oligomerization domain-like receptor protein 3 inflammasome activation contributing to the formation of calcium oxalate stone in HK-2 cells

Abstract

Objective To observe the mechanism of nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome to the formation of calcium oxalate stone after exposure to high concentrate of oxalate. Methods The human kidney proximal tubular epithelial HK-2 cells were cultured and stimulated with different concentrations of soluble oxalate cells. Lactate dehydrogenase (LDH) released assays from cell culture medium and 4’, 6-diamidino-2-phenylindole (DAPI) staining were used to determine the effect of cellular toxicity and injury after exposure to oxalate. All the cells of oxalate group and control group were incubated with calcium oxalate monohydrate (COM) crystals for 24 h, and cell surface adhesion of crystals were observed using an inverted phase-contrast microscope. Western blotting and real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) were applied to detect protein level and mRNA quantity of NLRP3, cysteinyl aspartate-specific protease-1 (Caspase-1), and interleukin-1β (IL-1β) from HK-2 cell lysates after exposure to oxalate. FQ-PCR was applied to analyse mRNA quantity of hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), hyaluronan synthase 3 (HAS3), osteopontin (OPN) and CD44 from HK-2 cell after exposure to oxalate. Intracellular reactive oxygen species (ROS) generation was estimated by the method of using 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) as per constructor’s protocol after exposure to oxalate. Thereafter, N-acetylcysteine (NAC), a scavenger of ROS was pre-treated with HK-2 cells for 2 h, and Western blotting and FQ-PCR were applied to detect protein level and mRNA quantity of NLRP3 gene. HK-2 cells were transfected by NLRP3-small interfering RNA (siRNA). After oxalate stimulation, an inverted phase-contrast microscope was used to detect the number of the binding COM crystals on the surface of the cells, and the levers of HAS1, HAS2, HAS3, OPN and CD44 mRNA through FQ-PCR. Results Exposure of HK-2 cells to different concentrations of oxalate for 24 h, it observed that exposure to the oxalate concentrations (0.8 mmol/L) led to significantly higher in LDH release than the oxalate concentrations (0.6 mmol/L) [(528.37±25.65) vs. (300.55±17.18) mmol/L, P=0.001], and the DAPI staining showed different cell number: 55.67±3.30, 52.33±6.13, 52.33±2.05, 58.00±9.09, 50.00±1.63, 47.33±2.05, 33.67±3.30, 20.33±2.05, and the result showed that exposure to the oxalate concentrations (1.0 mmol/L) led to a meaningful reduction in the number of cells compared with the oxalate concentrations (0.8 mmol/L) (33.67±4.04 vs. 47.33±2.52, P=0.008). Using an inverted phase-contrast microscope observed that a significantly greater number of the binding COM crystals on the surface of the high-oxalate-exposed cells as compared to the controls [(7.34±0.82)% vs. (5.29±0.73)%, P=0.001]. Exposure of HK-2 cells to high concentration oxalate for 24 h significantly increased the expression of protein (1.82±0.04 vs. 1.00±0.02, P=0.000; 1.62±0.23 vs. 1.00±0.06, P=0.021; 1.37±0.16 vs. 1.00±0.18, P=0.029) and mRNA levels (1.15±0.26 vs. 0.81±0.11, P=0.011; 2.16±0.29 vs. 1.04±0.14, P=0.009; 1.80±0.02 vs. 1.00±0.15, P=0.002) of NLRP3, Caspase-1, IL-1β when compared with the control, and the mRNA level (1.35±0.25 vs. 0.81±0.11, P=0.049; 2.46±0.29 vs. 0.84±0.14, P=0.002; 0.90±0.02 vs. 0.60±0.15, P=0.039; 1.68±0.27 vs. 1.00±0.22, P=0.049; 2.03±0.47 vs. 1.00±0.15, P=0.040) of HAS1, HAS2, HAS3, OPN and CD44 significantly up-regulated compared with the control. Exposure to high concentration of oxalate for 24 hours, the intracellular ROS levels of HK-2 were meaningfully increased (736.67±80.21 vs. 208.67±35.73) by the method of using DCFH-DA (P=0.001). Cells were pre-incubated with a ROS scavenger (NAC) for 2 h, followed by treatment with oxalate. According to the western blotting analysis of HK-2 cell lysates, the blockade of ROS by NAC resulted in decreased the protein levels (1.53±0.31 vs. 4.50±0.40)of NLRP3 gene in HK-2 cells (P=0.001), and that in the control and the NAC group were 0.90±0.10 and 1.00±0.26 respectively (P=0.288). The silencing of the NLRP3 gene obviously reduced the number of calcium oxalate crystals adhesion to cells [(1.87±0.21)% vs. (3.57±0.12)%, P=0.003], and the mRNA levels (1.06±0.07 vs. 2.44±0.47, P=0.015; 0.87±0.07 vs. 1.48±0.22, P=0.021; 1.24±0.16 vs. 1.72±0.10, P=0.026; 1.03±0.15 vs. 1.45±0.09, P=0.026; 0.63±0.07 vs. 1.31±0.12, P=0.002) of HAS1, HAS2, HAS3, OPN and CD44 mRNA were also descended. Conclusion Activation of the NLRP3 inflammasome induces by the generation of ROS in the formation of calcium oxalate kidney stone; The NLRP3 inflammasome activation engages by changing the renal tubular epithelial adhesion of crystal and involved in the formation of the stone. Key words: Nucleotide binding oligomerization domain-like receptor protein 3 inflammasome; Oxalate; Reactive oxygen species; Calcium oxalate stone; HK-2