| Objective To explore the mechanism of the formation of calcium oxalate kidney after activation of NLRP3 inflammatory body.Methods The expression of P38 protein was detected by immunohistochemistry in human calcium oxalate stones kidney tissues and normal renal tissues.Being cultured of NRK-52 E cells were treated with different concentrations of P38 MAPK pathway inhibitor(SB203580)(0,20,40,60,80,1.00 umol/L)and different concentrations of oxalate(0,0.2,0.4,0.6,0.8,1.0,mmol/L)for 24 h.The toxicity of oxalate and SB203580 to NRK-52 E cells was detected by MTT assay and LDH released assays from cell culture medium.All the cells of control group,oxalate group and oxalate supplemented with SB203580 group being incubated for 24 h,the expression of P38 protein was observed by immunofluorescence confocal microscopy,and cell surface adhesion of crystals were observed using an inverted phase-contrast microscope.The protein level and m RNA quantity of NLRP3,P38 and adhesion factor OPN were detected by Western boltting and RT-q PCR from the cells of control group,oxalate group and oxalate supplemented with SB203580 group being incubated for 24 h.Oxalating treating NRK-52 E cells for 24 h,The green fluorescent probes were used to detect mitochondrial inner NRK-52 E cells mitochondrial changes.The NLRP3 gene was silenced by transfection of NLRP3-Si RNA and then treated with oxalic acid for 24 h,Western boltting was applied to detect protein level of NLRP3,P38 and OPN protein.The RT-q PCR was applied to analyse m RNA quantity of OPN,HAS1,HAS2,HAS3,and CD44 from NRK-52 E cells being transfected by NLRP3 si RNA.The hyperoxaluric rat model was established by an ethylene glycol method.The changes of organelles in rat renal tubular epithelial cells were observed by perspective electron microscopy.Western blotting was used to detect the expression of NLRP3,P38 and OPN in renal tissue of rats.The expression of P38 protein in renal tissue of rats with kidney and control group was detected by immunohistochemistry.The expression of NLRP3,P38,OPN,HAS1,HAS2,HAS3,and CD44 m RNA were detected by RT-q PCR.SPSS 20.0 software was used to analyze the experimental data.Results Immunohistochemistry showed that the expression level of P38 protein in renal tissue of patients with calcium oxalate kidney stones was significantly higher than that in normal renal tissue specimens,and then the level of P38 MAPK pathway was involved in the formation of calcium oxalate kidney.The results showed that the content of LDH in the medium was higher than that of the oxalate at 0-0.6mmol / L(P <0.05)when the oxalate concentration was 0.8mmol / L.When the concentration of SB203580 was 60 umol / L for 48 h,the content of LDH in the medium was significantly higher than that in the concentration of SB203580 at 0-40 umol / L(P <0.05).The expression of P38 protein in oxalate group was significantly higher than that in control group by immunofluorescence confocal microscopy.Oxalate and oxalate added SB203580 treatment NRK-52 E cells 24 h,Western blotting showed that the expression of NLRP3,P38 and adhesion protein OPN in oxalate group was higher than that in control group(P <0.05).There was no significant increase in P38 and OPN protein(P> 0.05),but there was no significant increase in the expression of NLRP3 protein in the oxalate inhibitor group compared with the control group(P <0.05).Oxalate(0.6mmol / L)treatment of NRK-52 E cells 24 h,the use of mitochondrial green fluorescent probe detection found NRK-52 E intracellular mitochondria swelling.The expression of NLRP3 protein,P38 protein and adhesion factor OPN protein decreased(P <0.05)after 24 hours of oxalate treatment with NRK-52 E cells transfected with NLRP3-Si RNA.The expressions of CD44,HAS1,HAS2,HAS3 and OPN on the cell surface were significantly decreased(P <0.05).The calcium oxalate kidney stone model of SD rats was established by ethylene glycol drinking water method.The nuclei in the renal tubular epithelial cells of the rats with high oxalic acid were observed by means of perspective electron microscopy.The nuclei were oval-shaped.The expression of P38 protein in renal tissue of rats with high oxalic acid was significantly higher than that in the control group.Western blotting showed that the expression of NLRP3,P38 and OPN protein in renal tissue of rats was significantly higher than that of normal rat kidney(P <0.05).The m RNA expression of P38,NLRP3,Caspase-1,IL-1β,HAS1,HAS2,HAS3,CD44 and OPN in renal tissue of rats with high oxalate was also significantly higher than that of normal rat kidney(P <0.05).Conclusion Oxalate can cause renal tubular epithelial cells "cell coke death".P38 MAPK pathway involved in the formation of calcium oxalate kidney stones.Activation of NLRP3 Inflammatory Bodies affects the formation of renal tubular epithelial cells through the P38 MAPK pathway,which promotes the formation of kidney stones,thus providing a theoretical basis for the treatment and prevention of calcium oxalate stones. |
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