A Preliminary Study Of The NLRP3 Inflammasome Promoting The Formation Of Calcium Oxalate Stone

Objective To clear the role of the NLRP3 inflammasome activation in the formation and progression of calcium oxalate kidney stone, and to explore the mechanism of the NLRP3 inflammasome promoting the formation of calcium oxalate kidney stone.Methods The immunohistochemical detection was carried out to analyse the expression level of NLRP3, Caspase-1, and IL-1β in human calcium oxalate stones kidney tissues and normal renal tissues. HK-2 cells were cultured and stimulated with different concentrations of soluble oxalate(0,0.1, 0.2,0.4, 0.6, 0.8, 1.0, 2.0m M) cells for 24 h. LDH released assays from cell culture medium, DAPI staining, MTT assay and CCK-8 assay were used to determine the effect of cellular toxicity and viability after exposure to oxalate. All the cells of oxalate group and control group were incubated with COM crystals for 24 h, and cell surface adhesion of crystals were observed using an inverted phase-contrast microscope. Western boltting and RT-q PCR were applied to detect protein level and m RNA quantity of NLRP3,Caspase-1, and IL-1β from HK-2 cell lysates after exposure to oxalate. RT-q PCR was applied to analyse m RNA quantity of HAS1, HAS2, HAS3, OPN and CD44 from HK-2 cell after exposure to oxalate. Intracellular ROS generation was estimated by the method of using DCFH-DA as per constructor’s protocol after exposure to oxalate.Thereafter, NAC, a scavenger of ROS was pre-treated with HK-2 cells for 2h, and Western boltting and RT-q PCR were applied to detect protein level and m RNA quantity of NLRP3 gene. HK-2 cells were transfected by NLRP3-si RNA. 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 m RNA through RT-q PCR. We established an ethylene glycol method induced hyperoxaluric rat model featured by crystalline material within tubule lumens examined by HE staining, Pizzolato staining and polarizing microscope scanning. Western boltting was applied to detect protein level of NLRP3, Caspase-1, and IL-1β from tissue lysates in rat model, and RT-q PCR wereapplied to detect m RNA quantity of NLRP3, Caspase-1, IL-1β, HAS1, HAS2, HAS3,OPN and CD44. All the experimental results were summarized and analyzed by SPSS20.0 statistical analysis software.Results In renal tissue samples obtained from patients with calcium oxalate stone disease,we demonstrated that the expression level of NLRP3, Caspase-1, and IL-1β were above to the normal renal tissue samples, and these results suggested that the NLRP3 inflammasome activation may exist in the formation of calcium oxalate stone. LDH activity was increased significantly in 0.8m M oxalate group(P<0.05) after treatment with oxalate. Relying on DAPI dyeing, it showed that the number of cells was not meaningfully reduced until the concentration of oxalate up to 1.0m M(P<0.05). MTT assay and CCK-8 assay were then used to influence the viability of cells when the concentration of oxalic acid up to 1.0m M(P<0.05) after exposure to oxalate.Exposure of HK-2 cells to oxalate(0.8m M) for 24 h significantly increased the protein expression level of NLRP3, Caspase-1, IL-1β, and the m RNA level of NLRP3,Caspase-1, IL-1β, HA, CD44, and OPN(P<0.05), and even increased the calcium oxalate crystals adhesive ability(P<0.05). Exposure to high concentration of oxalate(0.8m M) for 24 hours, the intracellular ROS levels of HK-2 were meaningfully increased by the method of using DCFH-DA(P<0.05). Cells were pre-incubated with a ROS scavenger(NAC) for 2 h, followed by treatment with oxalate(0.8m M).According to the western blotting and RT-q PCR analysis of HK-2 cell lysates, the blockade of ROS by NAC resulted in decreased the protein levels of NLRP3 gene in HK-2 cells(P<0.05). The silencing of the NLRP3 gene obviously reduced the number of calcium oxalate crystals adhesion to cells(P<0.05), and the levels of HAS1, HAS2,HAS3, OPN and CD44 m RNA were also descended(P<0.05). We established a hyperoxaluric rat model charactered by crystalline material within tubule lumens examined by renal histology with HE staining, Pizzolato staining and polarizing microscope scanning. And we detected that the protein levels of NLRP3, Caspase-1and IL-1β were remarkably increased in the lysates from the kidneys of animal model(P<0.05), consistent with the m RNA expression levels in the animal kidneys(P<0.05).And the m RNA levels of HAS1, HAS2, HAS3, OPN and CD44 m RNA wereobviously increased(P<0.05). These results demonstrated that the NLRP3 inflammasome was activated in the formation of calcium oxalate stone in vivo experiment.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; At the same time, it provides a new clue and targets for the mechanism of calcium oxalate kidney stones and control.